WorldWideScience

Sample records for prv nucleic acid

  1. The Nucleic Acid Database.

    Science.gov (United States)

    Berman, Helen M; Westbrook, John; Feng, Zukang; Iype, Lisa; Schneider, Bohdan; Zardecki, Christine

    2002-06-01

    The Nucleic Acid Database was established in 1991 as a resource to assemble and distribute structural information about nucleic acids. Over the years, the NDB has developed generalized software for processing, archiving, querying and distributing structural data for nucleic acid-containing structures. The architecture and capabilities of the Nucleic Acid Database, as well as some of the research enabled by this resource, are presented in this article.

  2. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2003-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  3. Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    2002-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  4. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  5. Locked nucleic acid

    DEFF Research Database (Denmark)

    Jepsen, Jan Stenvang; Sørensen, Mads D; Wengel, Jesper

    2004-01-01

    Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing very high affinity and excellent specificity toward complementary DNA and RNA, and LNA oligonucleotides have been applied as antisense molecules both in vitro and in vivo. In this review, we briefly describe the basic physioc...

  6. Peptide Nucleic Acid Synthons

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  7. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds known as peptide nucleic acids, bind complementary DNA and RNA strands, and generally do so more strongly than the corresponding DNA or RNA strands while exhibiting increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from a...... a group consisting of naturally-occurring nucleobases and non-naturally-occurring nucleobases, including 2,6-diaminopurine, attached to a polyamide backbone, and contain alkyl amine side chains....

  8. Nucleic Acid-Based Nanoconstructs

    Science.gov (United States)

    Focuses on the design, synthesis, characterization, and development of spherical nucleic acid constructs as effective nanotherapeutic, single-entity agents for the treatment of glioblastoma multiforme and prostate cancers.

  9. Nucleic acid based molecular devices.

    Science.gov (United States)

    Krishnan, Yamuna; Simmel, Friedrich C

    2011-03-28

    In biology, nucleic acids are carriers of molecular information: DNA's base sequence stores and imparts genetic instructions, while RNA's sequence plays the role of a messenger and a regulator of gene expression. As biopolymers, nucleic acids also have exciting physicochemical properties, which can be rationally influenced by the base sequence in myriad ways. Consequently, in recent years nucleic acids have also become important building blocks for bottom-up nanotechnology: as molecules for the self-assembly of molecular nanostructures and also as a material for building machinelike nanodevices. In this Review we will cover the most important developments in this growing field of nucleic acid nanodevices. We also provide an overview of the biochemical and biophysical background of this field and the major "historical" influences that shaped its development. Particular emphasis is laid on DNA molecular motors, molecular robotics, molecular information processing, and applications of nucleic acid nanodevices in biology. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. [Circulating nucleic acids and infertility].

    Science.gov (United States)

    Scalici, E; Mullet, T; Ferrières Hoa, A; Gala, A; Loup, V; Anahory, T; Belloc, S; Hamamah, S

    2015-09-01

    Circulating nucleic acids (cell-free DNA and microRNAs) have for particularity to be easily detectable in the biological fluids of the body. Therefore, they constitute biomarkers of interest in female and male infertility care. Indeed, in female, they can be used to detect ovarian reserve disorders (polycystic ovary syndrome and low functional ovarian reserve) as well as to assess follicular microenvironment quality. Moreover, in men, their expression levels can vary in case of spermatogenesis abnormalities. Finally, circulating nucleic acids have also the ability to predict successfully the quality of in vitro embryo development. Their multiple contributions during assisted reproductive technology (ART) make of them biomarkers of interest, for the development of new diagnostic and/or prognostic tests, applied to our specialty. Circulating nucleic acids would so offer the possibility of personalized medical care for infertile couples in ART. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  11. Exosomes as nucleic acid nanocarriers.

    Science.gov (United States)

    van den Boorn, Jasper G; Dassler, Juliane; Coch, Christoph; Schlee, Martin; Hartmann, Gunther

    2013-03-01

    Exosomes are nano-sized vesicles produced naturally by many cell types. They are specifically loaded with nucleic acid cargo, dependent on the exosome-producing cell and its homeostatic state. As natural intercellular shuttles of miRNA, exosomes influence an array of developmental, physiological and pathological processes in the recipient cell or tissue to which they can be selectively targeted by their tetraspanin surface-domains. By a review of current research, we illustrate here why exosomes are ideal nanocarriers for use in the targeted in vivo delivery of nucleic acids. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Molecular Structure of Nucleic Acids

    Indian Academy of Sciences (India)

    A structure for nucleic acid has already been proposed by Pauling and Corey [1]. They kindly made'their manuscript available to us in advance of publication. Their model consists of three inter-twined chains, with the phosphates near the fibre axis, and the bases on the outside. In our opinion, this structure is unsatisfactory ...

  13. Histidine-Containing Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2000-01-01

    Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics.......Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics....

  14. Higher Order Structure and Binding of Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    Peptide nucleic acids and analogues of peptide nucleic acids are used to form duplex, triplex, and other structures with nucleic acids and to modify nucleic acids. The peptide nucleic acids and analogues thereof also are used to modulate protein activity through, for example, transcription arrest......, transcription initiation, and site specific cleavage of nucleic acids....

  15. Self-assembling multimeric nucleic acid constructs

    Science.gov (United States)

    Cantor, C.R.; Niemeyer, C.M.; Smith, C.L.; Sano, Takeshi; Hnatowich, D.J.; Rusckowski, M.

    1996-10-01

    The invention is directed to constructs and compositions containing multimeric forms of nucleic acid. Multimeric nucleic acids comprise single-stranded nucleic acids attached via biotin to streptavidin and bound with a functional group. These constructs can be utilized in vivo to treat or identify diseased tissue or cells. Repeated administrations of multimeric nucleic acid compositions produce a rapid and specific amplification of nucleic acid constructs and their attached functional groups. For treatment purposes, functional groups may be toxins, radioisotopes, genes or enzymes. Diagnostically, labeled multimeric constructs may be used to identify specific targets in vivo or in vitro. Multimeric nucleic acids may also be used in nanotechnology and to create self-assembling polymeric aggregates such as membranes of defined porosity, microcircuits and many other products. 5 figs.

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

    National Research Council Canada - National Science Library

    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...

  17. Uses of Nucleic Acid Analogues in the Inhibition of Nucleic Acid Amplification

    DEFF Research Database (Denmark)

    1999-01-01

    The present invention relates to the use of nucleic acid analogues in blocking nucleic acid ampli?cation procedures and to diagnostic and analytical techniques based thereon. Also included are kits for use in the conduct of nucleic acid ampli?cation reactions....

  18. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø

    2011-01-01

    Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing...... small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of aptamer selection, proteinaptamer recognition, protease inhibition, and advantages of aptamers...... for pharmacological intervention with pathophysiological functions of proteases. Aptamers can be selected so that they bind their targets highly specifically and with affinities corresponding to K(D) values in the nM range. Aptamers can be selected so that they recognize their targets conformation...

  19. Synthetic Procedures for Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  20. Double-Stranded Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2001-01-01

    A novel class of compounds, known as peptide nucleic acids, form double-stranded structures with one another and with ssDNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  1. The Nucleic Acid Database: Present and Future

    OpenAIRE

    Berman, Helen M.; Gelbin, Anke; Clowney, Lester; Hsieh, Shu-Hsin; Zardecki, Christine; Westbrook, John

    1996-01-01

    The Nucleic Acid Database is a relational database containing information about three-dimensional nucleic acid structures. The methods used for data processing, structure validation, database management and information retrieval, as well as the various services available via the World Wide Web, are described. Plans for the future include greater reliance on the Macromolecular Crystallographic Information File for both data processing and data management.

  2. An introduction to peptide nucleic acid

    DEFF Research Database (Denmark)

    Nielsen, P E; Egholm, M

    1999-01-01

    Peptide Nucleic Acid (PNA) is a powerful new biomolecular tool with a wide range of important applications. PNA mimics the behaviour of DNA and binds complementary nucleic acid strands. The unique chemical, physical and biological properties of PNA have been exploited to produce powerful biomolec...

  3. Methods for Identifying Ligands that Target Nucleic Acid Molecules and Nucleic Acid Structural Motifs

    Science.gov (United States)

    Disney, Matthew D. (Inventor); Childs-Disney, Jessica L. (Inventor)

    2017-01-01

    Disclosed are methods for identifying a nucleic acid (e.g., RNA, DNA, etc.) motif which interacts with a ligand. The method includes providing a plurality of ligands immobilized on a support, wherein each particular ligand is immobilized at a discrete location on the support; contacting the plurality of immobilized ligands with a nucleic acid motif library under conditions effective for one or more members of the nucleic acid motif library to bind with the immobilized ligands; and identifying members of the nucleic acid motif library that are bound to a particular immobilized ligand. Also disclosed are methods for selecting, from a plurality of candidate ligands, one or more ligands that have increased likelihood of binding to a nucleic acid molecule comprising a particular nucleic acid motif, as well as methods for identifying a nucleic acid which interacts with a ligand.

  4. An ultrasensitive photoelectrochemical nucleic acid biosensor

    Science.gov (United States)

    Gao, Zhiqiang; Tansil, Natalia C.

    2005-01-01

    A simple and ultrasensitive procedure for non-labeling detection of nucleic acids is described in this study. It is based on the photoelectrochemical detection of target nucleic acids by forming a nucleic acid/photoreporter adduct layer on an ITO electrode. The target nucleic acids were hybridized with immobilized oligonucleotide capture probes on the ITO electrode. A subsequent binding of a photoreporter—a photoactive threading bis-intercalator consisting of two N,N′-bis(3-propyl-imidazole)-1,4,5,8-naphthalene diimides (PIND) linked by a Ru(bpy)22+ (bpy = 2,2′-bipyridine) complex (PIND–Ru–PIND)—allowed for photoelectrochemical detection of the target nucleic acids. The extremely low dissociation rate of the adduct and the highly reversible photoelectrochemical response under visible light illumination (490 nm) make it possible to conduct nucleic acid detection, with a sensitivity enhancement of four orders of magnitude over voltammetry. These results demonstrate for the first time the potential of photoelectrochemical biosensors for PCR-free ultrasensitive detection of nucleic acids. PMID:16061935

  5. Nucleic acid diagnostic approaches in parasitology.

    Science.gov (United States)

    Kerttula, Anne-Marie; Lavikainen, Antti

    Nucleic acid diagnostic technologies are partly replacing traditional microscopy and antigen detection methods in parasitological diagnostics. In particular, the diagnostics of parasitic diarrhea is undergoing a transformation due to the application of polymerase chain reaction (PCR) tests. Diagnostics of malaria is still based on microscopy, but rapid nucleic acid tests are emerging. Laboratories of clinical microbiology in Finland currently provide PCR tests e.g. for intestinal protozoa, Toxoplasma and Trichomonas. Nucleic acid diagnostic methods are superior in specificity and sensitivity, but may give false positive results after a treated infection.

  6. Chip-based sequencing nucleic acids

    Science.gov (United States)

    Beer, Neil Reginald

    2014-08-26

    A system for fast DNA sequencing by amplification of genetic material within microreactors, denaturing, demulsifying, and then sequencing the material, while retaining it in a PCR/sequencing zone by a magnetic field. One embodiment includes sequencing nucleic acids on a microchip that includes a microchannel flow channel in the microchip. The nucleic acids are isolated and hybridized to magnetic nanoparticles or to magnetic polystyrene-coated beads. Microreactor droplets are formed in the microchannel flow channel. The microreactor droplets containing the nucleic acids and the magnetic nanoparticles are retained in a magnetic trap in the microchannel flow channel and sequenced.

  7. Halogen Bonding in Nucleic Acid Complexes.

    Science.gov (United States)

    Kolář, Michal H; Tabarrini, Oriana

    2017-11-09

    Halogen bonding (X-bonding) has attracted notable attention among noncovalent interactions. This highly directional attraction between a halogen atom and an electron donor has been exploited in knowledge-based drug design. A great deal of information has been gathered about X-bonds in protein-ligand complexes, as opposed to nucleic acid complexes. Here we provide a thorough analysis of nucleic acid complexes containing either halogenated building blocks or halogenated ligands. We analyzed close contacts between halogens and electron-rich moieties. The phosphate backbone oxygen is clearly the most common halogen acceptor. We identified 21 X-bonds within known structures of nucleic acid complexes. A vast majority of the X-bonds is formed by halogenated nucleobases, such as bromouridine, and feature excellent geometries. Noncovalent ligands have been found to form only interactions with suboptimal interaction geometries. Hence, the first X-bonded nucleic acid binder remains to be discovered.

  8. Nanoparticulate systems for nucleic acid delivery

    NARCIS (Netherlands)

    Varkouhi, A.K.

    2011-01-01

    Development of carrier systems with controllable physicochemical and delivery properties has opened up the possibility of nanomedicines containing nucleic acids. In the last decades, much effort has been dedicated to two exciting approaches in biomedicine, namely gene and RNA interference

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

    OpenAIRE

    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 f...

  10. Perfect Strangers: Inorganic Photochemistry and Nucleic Acids

    Science.gov (United States)

    Carter, Pamela J.; Ciftan, Suzanne A.; Sistare, Mark F.; Holden Thorp, H.

    1997-06-01

    The applications of inorganic photochemistry to nucleic acid chemistry are discussed. A brief review of nucleic acid structure is given. Methods for probing DNA using emissive inorganic complexes are discussed. Photoreactions that damage DNA by hydrogen atom transfer from sugar or electron abstraction from guanine are presented. The method of photochemical footprinting using a diplatinum photocatalyst is described. The final section discusses advances in combinatorial selection experiments that increase the urgency for rapid screening methods such as those derived from inorganic photochemistry.

  11. NPIDB: nucleic acid?protein interaction database

    OpenAIRE

    Kirsanov, Dmitry D.; Zanegina, Olga N.; Aksianov, Evgeniy A.; Spirin, Sergei A.; Karyagina, Anna S.; Alexeevski, Andrei V

    2012-01-01

    The Nucleic acid?Protein Interaction DataBase (http://npidb.belozersky.msu.ru/) contains information derived from structures of DNA?protein and RNA?protein complexes extracted from the Protein Data Bank (3846 complexes in October 2012). It provides a web interface and a set of tools for extracting biologically meaningful characteristics of nucleoprotein complexes. The content of the database is updated weekly. The current version of the Nucleic acid?Protein Interaction DataBase is an upgrade ...

  12. Carbohydrate Polymers for Nonviral Nucleic Acid Delivery

    OpenAIRE

    Sizovs, Antons; McLendon, Patrick M.; Srinivasachari, Sathya; Reineke, Theresa M.

    2010-01-01

    Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and ...

  13. Novel Biochip Platform for Nucleic Acid Analysis

    Directory of Open Access Journals (Sweden)

    Juan J. Diaz-Mochon

    2012-06-01

    Full Text Available This manuscript describes the use of a novel biochip platform for the rapid analysis/identification of nucleic acids, including DNA and microRNAs, with very high specificity. This approach combines a unique dynamic chemistry approach for nucleic acid testing and analysis developed by DestiNA Genomics with the STMicroelectronics In-Check platform, which comprises two microfluidic optimized and independent PCR reaction chambers, and a sequential microarray area for nucleic acid capture and identification by fluorescence. With its compact bench-top “footprint” requiring only a single technician to operate, the biochip system promises to transform and expand routine clinical diagnostic testing and screening for genetic diseases, cancers, drug toxicology and heart disease, as well as employment in the emerging companion diagnostics market.

  14. Imaging Functional Nucleic Acid Delivery to Skin.

    Science.gov (United States)

    Kaspar, Roger L; Hickerson, Robyn P; González-González, Emilio; Flores, Manuel A; Speaker, Tycho P; Rogers, Faye A; Milstone, Leonard M; Contag, Christopher H

    2016-01-01

    Monogenic skin diseases arise from well-defined single gene mutations, and in some cases a single point mutation. As the target cells are superficial, these diseases are ideally suited for treatment by nucleic acid-based therapies as well as monitoring through a variety of noninvasive imaging technologies. Despite the accessibility of the skin, there remain formidable barriers for functional delivery of nucleic acids to the target cells within the dermis and epidermis. These barriers include the stratum corneum and the layered structure of the skin, as well as more locally, the cellular, endosomal and nuclear membranes. A wide range of technologies for traversing these barriers has been described and moderate success has been reported for several approaches. The lessons learned from these studies include the need for combinations of approaches to facilitate nucleic acid delivery across these skin barriers and then functional delivery across the cellular and nuclear membranes for expression (e.g., reporter genes, DNA oligonucleotides or shRNA) or into the cytoplasm for regulation (e.g., siRNA, miRNA, antisense oligos). The tools for topical delivery that have been evaluated include chemical, physical and electrical methods, and the development and testing of each of these approaches has been greatly enabled by imaging tools. These techniques allow delivery and real time monitoring of reporter genes, therapeutic nucleic acids and also triplex nucleic acids for gene editing. Optical imaging is comprised of a number of modalities based on properties of light-tissue interaction (e.g., scattering, autofluorescence, and reflectance), the interaction of light with specific molecules (e.g., absorbtion, fluorescence), or enzymatic reactions that produce light (bioluminescence). Optical imaging technologies operate over a range of scales from macroscopic to microscopic and if necessary, nanoscopic, and thus can be used to assess nucleic acid delivery to organs, regions, cells

  15. Multifunctional Nucleic Acids for Tumor Cell Treatment

    DEFF Research Database (Denmark)

    Pofahl, Monika; Wengel, Jesper; Mayer, Günter

    2014-01-01

    -proliferative and antimiR function in one 37-nucleotide nucleic acid molecule. It inhibits cancer cell growth and induces gene expression that is pathologically damped by an oncomir. These findings will have a strong impact on future developments regarding aptamer- and antimiR-related applications for tumor targeting......We report on a multifunctional nucleic acid, termed AptamiR, composed of an aptamer domain and an antimiR domain. This composition mediates cell specific delivery of antimiR molecules for silencing of endogenous micro RNA. The introduced multifunctional molecule preserves cell targeting, anti...

  16. A locked nucleic Acid-based nanocrawler

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Pasternak, Karol; Campbell, Meghan A

    2013-01-01

    Herein we introduce a novel fluorescent LNA/DNA machine, a nanocrawler, which reversibly moves along a directionally polar complementary road controlled by affinity-enhancing locked nucleic acid (LNA) monomers and additional regulatory strands. Polyaromatic hydrocarbon (PAH) dyes attached to 2...

  17. Thermodynamic Analysis of Nylon Nucleic Acids

    Science.gov (United States)

    Liu, Yu; Wang, Risheng; Ding, Liang; Sha, Ruojie; Lukeman, Philip S.

    2010-01-01

    The stability and structure of nylon nucleic acid duplexes with complementary DNA and RNA strands was examined. Thermal denaturing studies of a series of oligonucleotides containing nylon nucleic acids (1 to 5 amide linkages) revealed that the amide linkage enhanced significantly the binding affinity of nylon nucleic acids towards both complementary DNA (up to 26°C increase in the thermal transition temperature (Tm) for 5 linkages) and RNA (around 15°C increase in Tm for 5 linkages) when compared with non-amide linked precursor strands. For both DNA and RNA complements, increasing derivatization decreased the melting temperatures of uncoupled molecules relative to unmodified strands; by contrast, increasing lengths of coupled copolymer raised Tm from less to slightly greater than Tm of unmodified strands. Thermodynamic data extracted from melting curves and CD spectra of nylon nucleic acid duplexes were consistent with loss of stability due to incorporation of pendent groups on the 2′ position of ribose, and recovery of stability upon linkage of the side chains. PMID:18543259

  18. Recent progress in nucleic acids isotachophoresis

    Czech Academy of Sciences Publication Activity Database

    Datinská, Vladimíra; Voráčová, Ivona; Schlecht, U.; Berka, J.; Foret, František

    2018-01-01

    Roč. 41, č. 1 (2018), s. 236-247 ISSN 1615-9306 R&D Projects: GA ČR(CZ) GBP206/12/G014 Institutional support: RVO:68081715 Keywords : isotachophoresis * nucleic acids * sample preparation Subject RIV: CB - Analytical Chemistry, Separation OBOR OECD: Analytical chemistry Impact factor: 2.557, year: 2016

  19. Locked and unlocked nucleosides in functional nucleic acids

    DEFF Research Database (Denmark)

    Doessing, Holger; Vester, Birte

    2011-01-01

    Nucleic acids are able to adopt a plethora of structures, many of which are of interest in therapeutics, bio- or nanotechnology. However, structural and biochemical stability is a major concern which has been addressed by incorporating a range of modifications and nucleoside derivatives. This rev....... This review summarizes the use of locked nucleic acid (LNA) and un-locked nucleic acid (UNA) monomers in functional nucleic acids such as aptamers, ribozymes, and DNAzymes....

  20. The Nucleic Acid Database: new features and capabilities

    OpenAIRE

    Coimbatore Narayanan, Buvaneswari; Westbrook, John; Ghosh, Saheli; Petrov, Anton I.; Sweeney, Blake; Zirbel, Craig L.; Leontis, Neocles B.; Berman, Helen M.

    2013-01-01

    The Nucleic Acid Database (NDB) (http://ndbserver.rutgers.edu) is a web portal providing access to information about 3D nucleic acid structures and their complexes. In addition to primary data, the NDB contains derived geometric data, classifications of structures and motifs, standards for describing nucleic acid features, as well as tools and software for the analysis of nucleic acids. A variety of search capabilities are available, as are many different types of reports. This article descri...

  1. Peptide Nucleic Acids Having Amino Acid Side Chains

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than the corresponding DNA or RNA strands, and exhibit increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from a group consisting of nat...... of naturally-occurring nucleobases and non-naturally-occurring nucleobases attached to a polyamide backbone, and contain alkylamine side chains....

  2. Digital Microfluidics for Nucleic Acid Amplification.

    Science.gov (United States)

    Coelho, Beatriz; Veigas, Bruno; Fortunato, Elvira; Martins, Rodrigo; Águas, Hugo; Igreja, Rui; Baptista, Pedro V

    2017-06-25

    Digital Microfluidics (DMF) has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

  3. Digital Microfluidics for Nucleic Acid Amplification

    Directory of Open Access Journals (Sweden)

    Beatriz Coelho

    2017-06-01

    Full Text Available Digital Microfluidics (DMF has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

  4. Nucleic acid compositions and the encoding proteins

    Science.gov (United States)

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  5. Spectrofluorimetric study of the binding of codeine to nucleic acids

    Science.gov (United States)

    Wang, Feng; Huang, Wei; Su, Liang; Dong, Zijia; Zhang, Shuai

    2009-06-01

    The characteristics of the interaction between codeine (CD) and nucleic acids were studied by ultraviolet-visible spectra and fluorescent spectra. It shows that there is a powerful ability in nucleic acids to quench the fluorescence intensity of codeine. The fluorescence quenching data were analyzed according to Stern-Volmer equation and Förster's nonradiative energy transfer mechanism. Thus the binding constant and the thermodynamic parameters between codeine and nucleic acids were obtained. The results show that codeine interacts with nucleic acids in a mode of groove binding and -OCH 3 of the codeine molecular combines with the groove of nucleic acids through hydrogen bond or van der Waals force.

  6. NAPP: the Nucleic Acid Phylogenetic Profile Database

    OpenAIRE

    Ott, Alban; Idali, Anouar; Marchais, Antonin; Gautheret, Daniel

    2017-01-01

    Nucleic acid phylogenetic profiling (NAPP) classifies coding and non-coding sequences in a genome according to their pattern of conservation across other genomes. This procedure efficiently distinguishes clusters of functional non-coding elements in bacteria, particularly small RNAs and cis-regulatory RNAs, from other conserved sequences. In contrast to other non-coding RNA detection pipelines, NAPP does not require the presence of conserved RNA secondary structure and therefore is likely to ...

  7. Prediction of protein and nucleic acid interactions

    OpenAIRE

    Cirillo, Davide

    2016-01-01

    The purpose of my doctoral studies has been the development of bioinformatics methods to quantitatively evaluate associations between proteins and nucleic acids (NAs). This thesis aims at providing insights into molecular features and still relatively unknown mechanisms of protein-NAs associations, such as RNA-binding proteins and long noncoding RNAs as well as transcription factors and regulatory DNA elements. In this work, I present two algorithms, catRAPID omics express and PAnDA, for the ...

  8. Carbohydrate Polymers for Nonviral Nucleic Acid Delivery

    Science.gov (United States)

    Sizovs, Antons; McLendon, Patrick M.; Srinivasachari, Sathya

    2014-01-01

    Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific delivery is also briefly reviewed. We contend that carbohydrates have contributed significantly to progress in the field of non-viral DNA delivery, and these new discoveries are impactful for developing new vehicles and materials for treatment of human disease. PMID:21504102

  9. Detection of nucleic acids by multiple sequential invasive cleavages

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Jeff G; Lyamichev, Victor I; Mast, Andrea L; Brow, Mary Ann D

    2012-10-16

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

  10. Use of Nucleic Acid Analogs for the Study of Nucleic Acid Interactions

    Directory of Open Access Journals (Sweden)

    Shu-ichi Nakano

    2011-01-01

    Full Text Available Unnatural nucleosides have been explored to expand the properties and the applications of oligonucleotides. This paper briefly summarizes nucleic acid analogs in which the base is modified or replaced by an unnatural stacking group for the study of nucleic acid interactions. We also describe the nucleoside analogs of a base pair-mimic structure that we have examined. Although the base pair-mimic nucleosides possess a simplified stacking moiety of a phenyl or naphthyl group, they can be used as a structural analog of Watson-Crick base pairs. Remarkably, they can adopt two different conformations responding to their interaction energies, and one of them is the stacking conformation of the nonpolar aromatic group causing the site-selective flipping of the opposite base in a DNA double helix. The base pair-mimic nucleosides can be used to study the mechanism responsible for the base stacking and the flipping of bases out of a nucleic acid duplex.

  11. Scaffolding along Nucleic Acid Duplexes Using 2'-Amino-Locked Nucleic Acids

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Wengel, Jesper

    2014-01-01

    Conspectus Incorporation of chemically modified nucleotide scaffolds into nucleic acids to form assemblies rich in function is an innovative area with great promise for nanotechnology and biomedical and material science applications. The intrinsic biorecognition potential of nucleic acids combined...... depends on the chemical nature of the modification, its price, its availability, and applications of the product. One of the most useful applications of the product LNA/DNA scaffolds containing 2'-amino-LNA is to detect complementary DNA and RNA targets. Examples of these applications include sensing...... functionalized 2'-amino-LNA scaffolds offer great opportunities for material science, diagnostics, and medicine of the future....

  12. Nucleic acid-based fluorescent probes and their analytical potential

    OpenAIRE

    Juskowiak, Bernard

    2010-01-01

    It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets...

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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. © The Author(s) 2016. Published by Oxford University Press.

  15. The Nucleic Acid Database: new features and capabilities.

    Science.gov (United States)

    Coimbatore Narayanan, Buvaneswari; Westbrook, John; Ghosh, Saheli; Petrov, Anton I; Sweeney, Blake; Zirbel, Craig L; Leontis, Neocles B; Berman, Helen M

    2014-01-01

    The Nucleic Acid Database (NDB) (http://ndbserver.rutgers.edu) is a web portal providing access to information about 3D nucleic acid structures and their complexes. In addition to primary data, the NDB contains derived geometric data, classifications of structures and motifs, standards for describing nucleic acid features, as well as tools and software for the analysis of nucleic acids. A variety of search capabilities are available, as are many different types of reports. This article describes the recent redesign of the NDB Web site with special emphasis on new RNA-derived data and annotations and their implementation and integration into the search capabilities.

  16. EGVI endoglucanase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel (Los Gatos, CA); Goedegebuur, Frits (Vlaardingen, NL); Ward, Michael (San Francisco, CA); Yao, Jian (Sunnyvale, CA)

    2010-10-05

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  17. EGVI endoglucanase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel (Los Gatos, CA); Goedegebuur, Frits (Vlaardingen, NL); Ward, Michael (San Francisco, CA); Yao, Jian (Sunnyvale, CA)

    2010-10-12

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  18. EGVI endoglucanase and nucleic acids encoding the same

    Science.gov (United States)

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2008-04-01

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  19. EGVII endoglucanase and nucleic acids encoding the same

    Science.gov (United States)

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2009-05-05

    The present invention provides an endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  20. EGVII endoglucanase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2012-02-14

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  1. Cytosolic nucleic acid sensors and innate immune regulation.

    Science.gov (United States)

    Ori, Daisuke; Murase, Motoya; Kawai, Taro

    2017-03-04

    During viral and bacterial infections, pathogen-derived cytosolic nucleic acids are recognized by the intracellular RNA sensors retinoic acid-inducible gene I and melanoma-differentiated gene 5 and intracellular DNA sensors, including cyclic-di-GMP-AMP synthase, absent in melanoma 2, interferon (IFN)-gamma inducible protein 16, polymerase III, and so on. Binding of intracellular nucleic acids to these sensors activates downstream signaling cascades, resulting in the production of type I IFNs and pro-inflammatory cytokines to induce appropriate systematic immune responses. While these sensors also recognize endogenous nucleic acids and activate immune responses, they can discriminate between self- and non-self-nucleic acids. However, dysfunction of these sensors or failure of regulatory mechanisms causes aberrant activation of immune response and autoimmune disorders. In this review, we focus on how intracellular immune sensors recognize exogenous nucleic acids and activate the innate immune system, and furthermore, how autoimmune diseases result from dysfunction of these sensors.

  2. Nucleic acid-binding glycoproteins which solubilize nucleic acids in dilute acid: re-examination of the Ustilago maydis glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Unrau, P.; Champ, D.R.; Young, J.L.; Grant, C.E.

    1980-01-01

    Holloman reported the isolation from Ustilago maydis of a glycoprotein which prevented the precipitation of nucleic acids in cold 5% trichloroacetic acid. Two glycoprotein fractions from U. maydis with this nucleic acid-solubilizing activity were isolated in our laboratory using improved purification procedures. The activity was not due to nuclease contamination. The glycoproteins are distinguished by: their ability to bind to concanavalin A-Sepharose; their differential binding to double- and single-stranded deoxyribonucleic acid, and to ribonucleic acid; their molecular weights (46,000 and 69,000); and the relative amounts present in growing versus nongrowing cells. Both fractions required sulfhydryl-reducing conditions for optimal yields, specific activity, and stability. Nucleic acid binding was cooperative, the minimum number of glycoproteins required to make a native T7 DNA molecule soluble in dilute acid being estimated at 2 and 15, respectively.

  3. Nucleic Acid Aptamers for Living Cell Analysis

    Science.gov (United States)

    Xiong, Xiangling; Lv, Yifan; Chen, Tao; Zhang, Xiaobing; Wang, Kemin; Tan, Weihong

    2014-06-01

    Cells as the building blocks of life determine the basic functions and properties of a living organism. Understanding the structure and components of a cell aids in the elucidation of its biological functions. Moreover, knowledge of the similarities and differences between diseased and healthy cells is essential to understanding pathological mechanisms, identifying diagnostic markers, and designing therapeutic molecules. However, monitoring the structures and activities of a living cell remains a challenging task in bioanalytical and life science research. To meet the requirements of this task, aptamers, as “chemical antibodies,” have become increasingly powerful tools for cellular analysis. This article reviews recent advances in the development of nucleic acid aptamers in the areas of cell membrane analysis, cell detection and isolation, real-time monitoring of cell secretion, and intracellular delivery and analysis with living cell models. Limitations of aptamers and possible solutions are also discussed.

  4. Electrical properties of nucleic acid bases

    Science.gov (United States)

    Basch, Harold; Garmer, D. R.; Jasien, P. G.; Krauss, M.; Stevens, W. J.

    1989-11-01

    The dipole polarizabilities for the five nucleic acid bases, uracil, cytosine, thymine, guanine, and adenine have been determined by the coupled perturbed Hartree-Fock (CPHF) method using a polarized double-zeta basis set. Electronic correlation corrections from second-order Møller-Plesset (MP2) perturbation theory are given. The treatment required to obtain accurate polarizabilities of these π systems was estimated from calculations on imidazole, benzene, and pyridine, giving results that are in good agreement with experiment. Similarly, optimal basis sets for static electrical properties were determined and applied to calculations of the dipole moments of the bases. With correlation corrections, these agree with experiment within 5% for all molecules except adenine.

  5. Intumescent features of nucleic acids and proteins

    Energy Technology Data Exchange (ETDEWEB)

    Alongi, Jenny, E-mail: jenny.alongi@polito.it; Cuttica, Fabio; Blasio, Alessandro Di; Carosio, Federico; Malucelli, Giulio

    2014-09-10

    Highlights: • The combustion resistance of DNA and caseins to different heat fluxes was studied. • Upon heating, DNA and caseins exhibited an intumescent behaviour. • The char derived from DNA was more stable and coherent than that from caseins. - Abstract: Are nucleic acids and proteins intumescent molecules? In order to get an answer, in the present manuscript, powders of deoxyribose nucleic acids (DNA) and caseins have been exposed to different heat fluxes under a cone calorimeter source and to the direct application of a propane flame. Under these conditions, DNA and caseins exhibited a typical intumescent behaviour, generating a coherent expanded cellular carbonaceous residue (char), extremely resistant to heat exposure. The resulting volumetric expansion as well as the resistance of the formed char turned out to be dependent on (i) the chemical structure of the chosen biomacromolecule, (ii) the evolution of ammonia and (iii) the adopted heat flux in cone calorimetry tests (namely, 25, 35, 50 and 75 kW/m{sup 2}). The presence of ribose units within the DNA backbone determined the formation of highly expanded and coherent residues as compared to those obtained from caseins. Indeed, under a heat flux of 35 kW/m{sup 2}, when a carbon source (i.e. common cane sugar) was added to caseins, the resulting char was similar to that formed by DNA. Furthermore, the char expansion was ascribed to the evolution of ammonia released by these biomacromolecules upon heating, as detected by thermogravimetry coupled to infrared spectroscopy, and confirmed by scanning electron microscopy experiments performed on the bubbles present in the residues of flammability tests.

  6. Unlocked nucleic acid - an RNA modification with broad potential

    DEFF Research Database (Denmark)

    Pasternak, Anna; Wengel, Jesper

    2011-01-01

    The first unlocked nucleic acid (UNA) monomer was described more than a decade ago, but only recent reports have revealed the true potential applications of this acyclic RNA mimic. UNA monomers enable the modulation of the thermodynamic stability of various nucleic acid structures such as RNA and...

  7. Nucleic acid drugs: a novel approach | Jarald | African Journal of ...

    African Journals Online (AJOL)

    Nucleic acid base sequence of proteins plays a crucial role in the expression of gene. The gene is responsible for the synthesis of proteins and these proteins, which are synthesized, are responsible for the biological process and also for dreadful diseases as well. Once if the nucleic acid sequence is altered, we would be ...

  8. Nucleic Acid Therapy: from humble beginnings a dynamic technology

    CSIR Research Space (South Africa)

    Millroy, L

    2011-02-01

    Full Text Available The term “nucleic acid therapy” encompasses a wide range of technologies for the treatment of a range of plant and animal ailments. As the name implies, it makes use of nucleic acid (either DNA or RNA) as a therapeutic agent. There are six branches...

  9. Nucleic Acid Amplification Testing in Suspected Child Sexual Abuse

    Science.gov (United States)

    Esernio-Jenssen, Debra; Barnes, Marilyn

    2011-01-01

    The American Academy of Pediatrics recommends that site-specific cultures be obtained, when indicated, for sexually victimized children. Nucleic acid amplification testing is a highly sensitive and specific methodology for identifying sexually transmitted infections. Nucleic acid amplification tests are also less invasive than culture, and this…

  10. MEANS AND METHODS FOR CLONING NUCLEIC ACID SEQUENCES

    NARCIS (Netherlands)

    Geertsma, Eric Robin; Poolman, Berend

    2008-01-01

    The invention provides means and methods for efficiently cloning nucleic acid sequences of interest in micro-organisms that are less amenable to conventional nucleic acid manipulations, as compared to, for instance, E.coli. The present invention enables high-throughput cloning (and, preferably,

  11. Gene Targeting and Expression Modulation by Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2010-01-01

    Peptide nucleic acids (PNA) are artificial structural mimics of nucleic acids capable of sequence specific hybridization to both RNA and DNA. Thus they have obvious potential as gene targeting agents for drug discovery approaches. An overview with emphasis on recent progress on RNA "interference"...

  12. Detecting Microbial Nucleic Acids within Nematode Bodies: A Photo Essay

    Science.gov (United States)

    We developed a taxa-specific, fluorescence in situ hybridization (FISH) technique to localize microbial nucleic acids within nematode bodies. This technique involves hybridization of a nucleic acid probe to target microbial sequences. Hybridization is detected microscopically, as the probes have f...

  13. Peptide Nucleic Acids Having Enhanced Binding Affinity and Sequence Specificity

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. Methods of increasing binding affinity and sequence specificity of peptide nucleic acids...

  14. Nucleic acid based fluorescent sensor for mercury detection

    Science.gov (United States)

    Lu, Yi; Liu, Juewen

    2013-02-05

    A nucleic acid enzyme comprises an oligonucleotide containing thymine bases. The nucleic acid enzyme is dependent on both Hg.sup.2+and a second ion as cofactors, to produce a product from a substrate. The substrate comprises a ribonucleotide, a deoxyribonucleotide, or both.

  15. Circulating nucleic acids damage DNA of healthy cells by integrating ...

    Indian Academy of Sciences (India)

    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 ...

  16. Use of Nucleic Acid Analogues in Diagnostics and Analytical Procedures

    DEFF Research Database (Denmark)

    2002-01-01

    Methods of capture, recognition, detection, identification or quantitation of nucleic acids and diagnostics uses generally are described in which are used: (a) a peptide nucleic acid (PNA) comprising a polyamide backbone bearing a plurality of ligands at respective spaced locations along said...

  17. Nucleic Acid Base Analog FRET-Pair Facilitating Detailed Structural Measurements in Nucleic Acid Containing Systems

    DEFF Research Database (Denmark)

    Börjesson, Karl; Preus, Søren; El-Sagheer, Afaf

    2009-01-01

    toward detailed studies of the inherent dynamics of nucleic acid structures. Moreover, the placement of FRET-pair chromophores inside the base stack will be a great advantage in studies where other (biomacro)molecules interact with the nucleic acid. Lastly, our study gives possibly the first truly solid...... distances covering up to more than one turn of the DNA duplex. Importantly, we show that the rigid stacking of the two base analogs, and consequently excellent control of their exact positions and orientations, results in a high control of the orientation factor and hence very distinct FRET changes...... as the number of bases separating tCO and tC(nitro) is varied. A set of DNA strands containing the FRET-pair at wisely chosen locations will, thus, make it possible to accurately distinguish distance- from orientation-changes using FRET. In combination with the good nucleobase analog properties, this points...

  18. Quantitative thermodynamic predication of interactions between nucleic acid and non-nucleic acid species using Microsoft excel.

    Science.gov (United States)

    Zou, Jiaqi; Li, Na

    2013-09-01

    Proper design of nucleic acid sequences is crucial for many applications. We have previously established a thermodynamics-based quantitative model to help design aptamer-based nucleic acid probes by predicting equilibrium concentrations of all interacting species. To facilitate customization of this thermodynamic model for different applications, here we present a generic and easy-to-use platform to implement the algorithm of the model with Microsoft(®) Excel formulas and VBA (Visual Basic for Applications) macros. Two Excel spreadsheets have been developed: one for the applications involving only nucleic acid species, the other for the applications involving both nucleic acid and non-nucleic acid species. The spreadsheets take the nucleic acid sequences and the initial concentrations of all species as input, guide the user to retrieve the necessary thermodynamic constants, and finally calculate equilibrium concentrations for all species in various bound and unbound conformations. The validity of both spreadsheets has been verified by comparing the modeling results with the experimental results on nucleic acid sequences reported in the literature. This Excel-based platform described here will allow biomedical researchers to rationalize the sequence design of nucleic acid probes using the thermodynamics-based modeling even without relevant theoretical and computational skills. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. Nucleic acid-binding polymers as anti-inflammatory agents

    Science.gov (United States)

    Lee, Jaewoo; Sohn, Jang Wook; Zhang, Ying; Leong, Kam W.; Pisetsky, David; Sullenger, Bruce A.

    2011-01-01

    Dead and dying cells release nucleic acids. These extracellular RNAs and DNAs can be taken up by inflammatory cells and activate multiple nucleic acid-sensing toll-like receptors (TLR3, 7, 8, and 9). The inappropriate activation of these TLRs can engender a variety of inflammatory and autoimmune diseases. The redundancy of the TLR family encouraged us to seek materials that can neutralize the proinflammatory effects of any nucleic acid regardless of its sequence, structure or chemistry. Herein we demonstrate that certain nucleic acid-binding polymers can inhibit activation of all nucleic acid-sensing TLRs irrespective of whether they recognize ssRNA, dsRNA or hypomethylated DNA. Furthermore, systemic administration of such polymers can prevent fatal liver injury engendered by proinflammatory nucleic acids in an acute toxic shock model in mice. Therefore these polymers represent a novel class of anti-inflammatory agent that can act as molecular scavengers to neutralize the proinflammatory effects of various nucleic acids. PMID:21844380

  20. Phosphorus SAD Phasing for Nucleic Acid Structures: Limitations and Potential

    Directory of Open Access Journals (Sweden)

    Joel M. Harp

    2016-10-01

    Full Text Available Phasing of nucleic acid crystal diffraction data using the anomalous signal of phosphorus, P-SAD, at Cukα wavelength has been previously demonstrated using Z-DNA. Since the original work on P-SAD with Z-DNA there has been, with a notable exception, a conspicuous absence of applications of the technique to additional nucleic acid crystal structures. We have reproduced the P-SAD phasing of Z-DNA using a rotating-anode source and have attempted to phase a variety of nucleic acid crystals using P-SAD without success. A comparison of P-SAD using Z-DNA and a representative nucleic acid, the Dickerson-Drew dodecamer, is presented along with a S-SAD using only two sulfurs to phase a 2’-thio modified DNA decamer. A theoretical explanation for the limitation of P-SAD applied to nucleic acids is presented to show that the relatively high atomic displacement parameter of phosphorus in the nucleic acid backbone is responsible for the lack of success in applying P-SAD to nucleic acid diffraction data.

  1. Fluorescent hybridization probes for nucleic acid detection.

    Science.gov (United States)

    Guo, Jia; Ju, Jingyue; Turro, Nicholas J

    2012-04-01

    Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.

  2. Continuously Tunable Nucleic Acid Hybridization Probes

    Science.gov (United States)

    Wu, Lucia R.; Wang, J. Sherry; Fang, John Z.; Reiser, Emily; Pinto, Alessandro; Pekker, Irena; Boykin, Richard; Ngouenet, Celine; Webster, Philippa J.; Beechem, Joseph; Zhang, David Yu

    2015-01-01

    In silico designed nucleic acid probes and primers often fail to achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. Here, we present a novel, on-the-fly method of tuning probe affinity and selectivity via the stoichiometry of auxiliary species, allowing independent and decoupled adjustment of hybridization yield for different probes in multiplexed assays. Using this method, we achieve near-continuous tuning of probe effective free energy (0.03 kcal·mol−1 granularity). As applications, we enforced uniform capture efficiency of 31 DNA molecules (GC content 0% – 100%), maximized signal difference for 11 pairs of single nucleotide variants, and performed tunable hybrid-capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples (FFPE). PMID:26480474

  3. NAPP: the Nucleic Acid Phylogenetic Profile Database.

    Science.gov (United States)

    Ott, Alban; Idali, Anouar; Marchais, Antonin; Gautheret, Daniel

    2012-01-01

    Nucleic acid phylogenetic profiling (NAPP) classifies coding and non-coding sequences in a genome according to their pattern of conservation across other genomes. This procedure efficiently distinguishes clusters of functional non-coding elements in bacteria, particularly small RNAs and cis-regulatory RNAs, from other conserved sequences. In contrast to other non-coding RNA detection pipelines, NAPP does not require the presence of conserved RNA secondary structure and therefore is likely to identify previously undetected RNA genes or elements. Furthermore, as NAPP clusters contain both coding and non-coding sequences with similar occurrence profiles, they can be analyzed under a functional perspective. We recently improved the NAPP pipeline and applied it to a collection of 949 bacterial and 68 archaeal species. The database and web interface available at http://napp.u-psud.fr/ enable detailed analysis of NAPP clusters enriched in non-coding RNAs, graphical display of phylogenetic profiles, visualization of predicted RNAs in their genome context and extraction of predicted RNAs for use with genome browsers or other software.

  4. Lateral flow biosensors for the detection of nucleic acid.

    Science.gov (United States)

    Zeng, Lingwen; Lie, Puchang; Fang, Zhiyuan; Xiao, Zhuo

    2013-01-01

    The detection of nucleic acid is of central importance for the diagnosis of genetic diseases, infectious agents, and biowarfare agents. Traditional strategies and technologies for nucleic acid detection are time-consuming and labor-intensive. Recently, isothermal strand-displacement reaction-based lateral flow biosensors have attracted a great deal of research interest because they are sensitive, simple, fast, and easy to use. Here, we describe a lateral flow biosensor based on isothermal strand-displacement polymerase reaction and gold nanoparticles for the visual detection of nucleic acid.

  5. Nucleic acid detection system and method for detecting influenza

    Science.gov (United States)

    Cai, Hong; Song, Jian

    2015-03-17

    The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

  6. Boronic acid-based autoligation of nucleic acids

    DEFF Research Database (Denmark)

    Barbeyron, R.; Vasseur, J.-J.; Smietana, M.

    2013-01-01

    Abstract: The development of synthetic systems displaying dynamic and adaptive characteristics is a formidable challenge with wide applications from biotechnology to therapeutics. Recently, we described a dynamic and programmable nucleic acid-based system relying on the formation of reversible...... boronate internucleosidic linkages. The DNA- or RNA-templated system comprises a 5′-ended boronic acid probe connecting a 3′-ended ribonucleosidic oligonucleotide partner. To explore the dominant factors that control the reversible linkage, we synthesized a series of 3′-end modified ribonucleotidic strands...

  7. Symmetry in nucleic acid structure and its role in protein--nucleic acid interactions

    Energy Technology Data Exchange (ETDEWEB)

    Sobell, H.M.

    1976-01-01

    It is clear that symmetry concepts will play an increasingly important role in understanding the structural aspects of many protein--nucleic acid interactions. This review summarizes current data and concepts along these lines. Exact crystallographic symmetries are rarely expected to occur in biological systems, and the departures from symmetry that occur may eventually prove to play important roles in modulating physiological function. In this respect, x-ray crystallography can make important additions to our understanding of the atomic nature of these associations, and it is hoped that the next review in this area will describe these.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    Two G-quadruplex forming sequences, 50-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4......-(1-pyrenylethynyl)phenylmethyl]glycerol (twisted intercalating nucleic acid, TINA). Incorporation of LNA or INA/TINA monomers provide as much as 8-fold improvement of anti-HIV-1 activity. We demonstrate for the first time a detailed analysis of the effect the incorporation of INA/TINA monomers in quadruplex forming...

  9. Assessment for Melting Temperature Measurement of Nucleic Acid by HRM

    National Research Council Canada - National Science Library

    Jing Wang; Xiaoming Pan; Xingguo Liang

    2016-01-01

    High resolution melting (HRM), with a high sensitivity to distinguish the nucleic acid species with small variations, has been widely applied in the mutation scanning, methylation analysis, and genotyping...

  10. Nanopore biosensors for detection of proteins and nucleic acids

    NARCIS (Netherlands)

    Maglia, Giovanni; Soskine, Mikhael

    2014-01-01

    Described herein are nanopore biosensors based on a modified cytolysin protein. The nanopore biosensors accommodate macromoiecules including proteins and nucleic acids, and may additionally comprise ligands with selective binding properties.

  11. NPIDB: Nucleic acid-Protein Interaction DataBase

    National Research Council Canada - National Science Library

    Kirsanov, Dmitry D; Zanegina, Olga N; Aksianov, Evgeniy A; Spirin, Sergei A; Karyagina, Anna S; Alexeevski, Andrei V

    2013-01-01

    The Nucleic acid-Protein Interaction DataBase (http://npidb.belozersky.msu.ru/) contains information derived from structures of DNA-protein and RNA-protein complexes extracted from the Protein Data Bank...

  12. Biological activity and biotechnological aspects of locked nucleic acids

    DEFF Research Database (Denmark)

    Lundin, Karin E; Højland, Torben; Hansen, Bo

    2013-01-01

    Locked nucleic acid (LNA) is one of the most promising new nucleic acid analogues that has been produced under the past two decades. In this chapter, we have tried to cover many of the different areas, where this molecule has been used to improve the function of synthetic oligonucleotides (ONs). ...... and the adaptation of enzymes for LNA incorporation are reviewed. Such enzymes may become important for the development of stabilized LNA-containing aptamers....

  13. Peptide Nucleic Acids Having 2,6-Diaminopurine Nucleobases

    DEFF Research Database (Denmark)

    2003-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. The peptide nucleic acids of the invention comprise ligands selected from a group cons...... consisting of naturally-occurring nucleobases and non-naturally-occurring nucleobases attached to a polyamide backbone. Some PNAs of the invention also contain C1-C8 alkylamine side chains....

  14. Nucleic acid-functionalized transition metal nanosheets for biosensing applications.

    Science.gov (United States)

    Mo, Liuting; Li, Juan; Liu, Qiaoling; Qiu, Liping; Tan, Weihong

    2017-03-15

    In clinical diagnostics, as well as food and environmental safety practices, biosensors are powerful tools for monitoring biological or biochemical processes. Two-dimensional (2D) transition metal nanomaterials, including transition metal chalcogenides (TMCs) and transition metal oxides (TMOs), are receiving growing interest for their use in biosensing applications based on such unique properties as high surface area and fluorescence quenching abilities. Meanwhile, nucleic acid probes based on Watson-Crick base-pairing rules are also being widely applied in biosensing based on their excellent recognition capability. In particular, the emergence of functional nucleic acids in the 1980s, especially aptamers, has substantially extended the recognition capability of nucleic acids to various targets, ranging from small organic molecules and metal ions to proteins and cells. Based on π-π stacking interaction between transition metal nanosheets and nucleic acids, biosensing systems can be easily assembled. Therefore, the combination of 2D transition metal nanomaterials and nucleic acids brings intriguing opportunities in bioanalysis and biomedicine. In this review, we summarize recent advances of nucleic acid-functionalized transition metal nanosheets in biosensing applications. The structure and properties of 2D transition metal nanomaterials are first discussed, emphasizing the interaction between transition metal nanosheets and nucleic acids. Then, the applications of nucleic acid-functionalized transition metal nanosheet-based biosensors are discussed in the context of different signal transducing mechanisms, including optical and electrochemical approaches. Finally, we provide our perspectives on the current challenges and opportunities in this promising field. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Highly fluorescent conjugated pyrenes in nucleic acid probes: (phenylethynyl)pyrenecarbonyl-functionalized locked nucleic acids.

    Science.gov (United States)

    Astakhova, Irina V; Korshun, Vladimir A; Wengel, Jesper

    2008-01-01

    In recent years, fluorescently labeled oligonucleotides have become a widely used tool in diagnostics, DNA sequencing, and nanotechnology. The recently developed (phenylethynyl)pyrenes are attractive dyes for nucleic acid labeling, with the advantages of long-wave emission relative to the parent pyrene, high fluorescence quantum yields, and the ability to form excimers. Herein, the synthesis of six (phenylethynyl)pyrene-functionalized locked nucleic acid (LNA) monomers M(1)-M(6) and their incorporation into DNA oligomers is described. Multilabeled duplexes display higher thermal stabilities than singly modified analogues. An increase in the number of phenylethynyl substituents attached to the pyrene results in decreased binding affinity towards complementary DNA and RNA and remarkable bathochromic shifts of absorption/emission maxima relative to the parent pyrene fluorochrome. This bathochromic shift leads to the bright fluorescence colors of the probes, which differ drastically from the blue emission of unsubstituted pyrene. The formation of intra- and interstrand excimers was observed for duplexes that have monomers M(1)-M(6) in both complementary strands and in numerous single-stranded probes. If more phenylethynyl groups are inserted, the detected excimer signals become more intense. In addition, (phenylethynyl)pyrenecarbonyl-LNA monomers M(4), M(5), and M(6) proved highly useful for the detection of single mismatches in DNA/RNA targets.

  16. Nucleic acid in-situ hybridization detection of infectious agents

    Science.gov (United States)

    Thompson, Curtis T.

    2000-04-01

    Limitations of traditional culture methods and newer polymerase chain reaction (PCR)-based methods for detection and speciation of infectious agents demonstrate the need for more rapid and better diagnostics. Nucleic acid hybridization is a detection technology that has gained wide acceptance in cancer and prenatal cytogenetics. Using a modification of the nucleic acid hybridization technique known as fluorescence in-situ hybridization, infectious agents can be detected in a variety of specimens with high sensitivity and specificity. The specimens derive from all types of human and animal sources including body fluids, tissue aspirates and biopsy material. Nucleic acid hybridization can be performed in less than one hour. The result can be interpreted either using traditional fluorescence microscopy or automated platforms such as micro arrays. This paper demonstrates proof of concept for nucleic acid hybridization detection of different infectious agents. Interpretation within a cytologic and histologic context is possible with fluorescence microscopic analysis, thereby providing confirmatory evidence of hybridization. With careful probe selection, nucleic acid hybridization promises to be a highly sensitive and specific practical diagnostic alternative to culture, traditional staining methods, immunohistochemistry and complicated nucleic acid amplification tests.

  17. Multiple sequence alignments of partially coding nucleic acid sequences

    Directory of Open Access Journals (Sweden)

    Fried Claudia

    2005-06-01

    Full Text Available Abstract Background High quality sequence alignments of RNA and DNA sequences are an important prerequisite for the comparative analysis of genomic sequence data. Nucleic acid sequences, however, exhibit a much larger sequence heterogeneity compared to their encoded protein sequences due to the redundancy of the genetic code. It is desirable, therefore, to make use of the amino acid sequence when aligning coding nucleic acid sequences. In many cases, however, only a part of the sequence of interest is translated. On the other hand, overlapping reading frames may encode multiple alternative proteins, possibly with intermittent non-coding parts. Examples are, in particular, RNA virus genomes. Results The standard scoring scheme for nucleic acid alignments can be extended to incorporate simultaneously information on translation products in one or more reading frames. Here we present a multiple alignment tool, codaln, that implements a combined nucleic acid plus amino acid scoring model for pairwise and progressive multiple alignments that allows arbitrary weighting for almost all scoring parameters. Resource requirements of codaln are comparable with those of standard tools such as ClustalW. Conclusion We demonstrate the applicability of codaln to various biologically relevant types of sequences (bacteriophage Levivirus and Vertebrate Hox clusters and show that the combination of nucleic acid and amino acid sequence information leads to improved alignments. These, in turn, increase the performance of analysis tools that depend strictly on good input alignments such as methods for detecting conserved RNA secondary structure elements.

  18. Analyzing and Building Nucleic Acid Structures with 3DNA

    OpenAIRE

    Colasanti, Andrew V.; Lu, Xiang-Jun; Olson, Wilma K.

    2013-01-01

    The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic...

  19. Extraction of viral nucleic acids: comparison of five automated nucleic acid extraction platforms.

    Science.gov (United States)

    Verheyen, Jens; Kaiser, Rolf; Bozic, Michael; Timmen-Wego, Monika; Maier, Barbara K; Kessler, Harald H

    2012-07-01

    Nucleic acid extraction has a major impact on the reliability of results in routine molecular diagnostics. Optimal isolation of nucleic acids and removal of inhibitors are essential. This study compares five different automated extraction platforms for the extraction of norovirus RNA from stool and cytomegalovirus (CMV) DNA from plasma samples. Norovirus positive stool samples and CMV positive plasma samples were aliquoted and analyzed using five different automated platforms (easyMAG, bioMerieux; m2000sp, Abbott; MagNA Pure LC 2.0, Roche; QiaSymphony, Qiagen; sample preparation module of the VERSANT kPCR Molecular System, Siemens). Similar sample input and output volumes, the identical real-time PCR cycler, and the identical assays for amplification and detection for norovirus RNA and CMV DNA, respectively, were chosen. Of 39 stool samples, 36 tested positive for norovirus RNA with all extraction platforms. The three discrepant samples showed inhibition after extraction with at least one platform. Only with the VERSANT platform all samples tested positive for both the target RNA and the internal controls. Of 42 plasma samples, 27 gave quantifiable results for CMV DNA with all extraction platforms. There was significant variance between viral concentrations when different extraction platforms were compared. The majority of the 15 discrepant samples showed low viral concentrations. The internal control of the CMV assay gave positive results for all samples tested below the limit of quantification. The five automated extraction platforms yielded comparable results. However, the extraction performance was found to be impaired by inhibitory substances in stool samples. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Molecular dynamics simulation of nucleic acids: successes, limitations, and promise.

    Science.gov (United States)

    Cheatham, T E; Young, M A

    In the last five years we have witnessed a significant increase in the number publications describing accurate and reliable all-atom molecular dynamics simulations of nucleic acids. This increase has been facilitated by the development of fast and efficient methods for treating the long-range electrostatic interactions, the availability of faster parallel computers, and the development of well-validated empirical molecular mechanical force fields. With these technologies, it has been demonstrated that simulation is not only capable of consistently reproducing experimental observations of sequence specific fine structure of DNA, but also can give detailed insight into prevalent problems in nucleic acid structure, ion association and specific hydration of nucleic acids, polyadenine tract bending, and the subtle environmental dependence of the A-DNA-B-DNA duplex equilibrium. Despite the advances, there are still issues with the methods that need to be resolved through rigorous controlled testing. In general, these relate to deficiencies of the underlying molecular mechanical potentials or applied methods (such as the imposition of true periodicity in Ewald simulations and the need for energy conservation), and significant limits in effective conformational sampling. In this perspective, we provide an overview of our experiences, provide some cautionary notes, and provide recommendations for further study in molecular dynamics simulation of nucleic acids. Copyright 2001 John Wiley & Sons, Inc. Biopolymers (Nucleic Acid Sci) 56: 232-256, 2001

  1. Peptide nucleic acids and their potential applications in biotechnology

    DEFF Research Database (Denmark)

    Buchardt, O.; Egholm, M.; Berg, R.H.

    1993-01-01

    Peptide nucleic acids (PNAs) are novel DNA mimics in which the sugar-phosphate backbone has been replaced with a backbone based on amino acids1-3. PNAs exhibit sequence-specific binding to DNA and RNA with higher affinities and specificities than unmodified DNA. They,are resistant to nuclease...

  2. Inorganic nanoparticles as nucleic acid transporters into eukaryotic cells

    Science.gov (United States)

    Amirkhanov, R. N.; Zarytova, V. F.; Zenkova, M. A.

    2017-02-01

    The review is concerned with inorganic nanoparticles (gold, titanium dioxide, silica, iron oxides, calcium phosphate) used as nucleic acid transporters into mammalian cells. Methods for the synthesis of nanoparticles and approaches to surface modification through covalent or noncovalent attachment of low- or high-molecular-weight compounds are considered. The data available from the literature on biological action of nucleic acids delivered into the cells by nanoparticles and on the effect of nanoparticles and their conjugates and complexes on the cell survival are summarized. Pathways of cellular internalization of nanoparticles and the mechanism of their excretion, as well as the ways of release of nucleic acids from their complexes with nanoparticles after the cellular uptake are described. The bibliography includes 161 references.

  3. Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology.

    Science.gov (United States)

    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. Copyright © 2011 Wiley Periodicals, Inc.

  4. Amino-containing magnetic nanoemulsions: elaboration and nucleic acid extraction

    Energy Technology Data Exchange (ETDEWEB)

    Veyret, Raphael [Unite Mixte CNRS-bioMerieux-2714, Ecole Normale Superieure de Lyon, 46 allee d' Italie, 69364 Lyon (France); Delair, Thierry [Unite Mixte CNRS-bioMerieux-2714, Ecole Normale Superieure de Lyon, 46 allee d' Italie, 69364 Lyon (France); Pichot, Christian [Unite Mixte CNRS-bioMerieux-2714, Ecole Normale Superieure de Lyon, 46 allee d' Italie, 69364 Lyon (France); Elaissari, Abdelhamid [Unite Mixte CNRS-bioMerieux-2714, Ecole Normale Superieure de Lyon, 46 allee d' Italie, 69364 Lyon (France)]. E-mail: hamid.elaissari@ens-lyon.fr

    2005-08-15

    Amino-containing magnetic colloids were prepared from highly magnetic oil-in-water (O/W) emulsions. The functionalization was performed by controlling the adsorption of polyethyleneimine onto negatively charged magnetic emulsions. The cationic magnetic nanodroplets were characterized in terms of chemical composition, particle size, size distribution, zeta potential and colloidal stability as a function of storage time. These amino-containing magnetic emulsions were assessed as a new tool for nucleic acid extraction and amplification. The adsorption of nucleic acids was mostly controlled by attractive electrostatic interactions. The adsorption efficiency of a model RNA was found to be encouraging and the captured nucleic acid molecules were directly enzymatically amplified in the presence of the magnetic particles without any elution step.

  5. Soni-removal of nucleic acids from inclusion bodies.

    Science.gov (United States)

    Neerathilingam, Muniasamy; Mysore, Sumukh; Gandham, Sai Hari A

    2014-05-23

    Inclusion bodies (IBs) are commonly formed in Escherichia coli due to over expression of recombinant proteins in non-native state. Isolation, denaturation and refolding of these IBs is generally performed to obtain functional protein. However, during this process IBs tend to form non-specific interactions with sheared nucleic acids from the genome, thus getting carried over into downstream processes. This may hinder the refolding of IBs into their native state. To circumvent this, we demonstrate a methodology termed soni-removal which involves disruption of nucleic acid-inclusion body interaction using sonication; followed by solvent based separation. As opposed to conventional techniques that use enzymes and column-based separations, soni-removal is a cost effective alternative for complete elimination of buried and/or strongly bound short nucleic acid contaminants from IBs. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Biomimetic High Density Lipoprotein Nanoparticles For Nucleic Acid Delivery

    Science.gov (United States)

    McMahon, Kaylin M.; Mutharasan, R. Kannan; Tripathy, Sushant; Veliceasa, Dorina; Bobeica, Mariana; Shumaker, Dale K.; Luthi, Andrea J.; Helfand, Brian T.; Ardehali, Hossein; Mirkin, Chad A.; Volpert, Olga; Thaxton, C. Shad

    2014-01-01

    We report a gold nanoparticle-templated high density lipoprotein (HDL AuNP) platform for gene therapy which combines lipid-based nucleic acid transfection strategies with HDL biomimicry. For proof-of-concept, HDL AuNPs are shown to adsorb antisense cholesterylated DNA. The conjugates are internalized by human cells, can be tracked within cells using transmission electron microscopy (TEM), and regulate target gene expression. Overall, the ability to directly image the AuNP core within cells, the chemical tailorability of the HDL AuNP platform, and the potential for cell-specific targeting afforded by HDL biomimicry make this platform appealing for nucleic acid delivery. PMID:21319839

  7. Recent Advances in Chemical Modification of Peptide Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Eriks Rozners

    2012-01-01

    Full Text Available Peptide nucleic acid (PNA has become an extremely powerful tool in chemistry and biology. Although PNA recognizes single-stranded nucleic acids with exceptionally high affinity and sequence selectivity, there is considerable ongoing effort to further improve properties of PNA for both fundamental science and practical applications. The present paper discusses selected recent studies that improve on cellular uptake and binding of PNA to double-stranded DNA and RNA. The focus is on chemical modifications of PNA's backbone and heterocyclic nucleobases. The paper selects representative recent studies and does not attempt to provide comprehensive coverage of the broad and vibrant field of PNA modification.

  8. Methods of introducing nucleic acids into cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Lajoie, Marc J.; Gregg, Christopher J.; Mosberg, Joshua A.; Church, George M.

    2017-06-27

    A method of introducing a nucleic acid sequence into a cell is provided where the cell has impaired or inhibited or disrupted DnaG primase activity or impaired or inhibited or disrupted DnaB helicase activity, or larger or increased gaps or distance between Okazaki fragments or lowered or reduced frequency of Okazaki fragment initiation, or the cell has increased single stranded DNA (ssDNA) on the lagging strand of the replication fork including transforming the cell through recombination with a nucleic acid oligomer.

  9. Enhancing and targeting nucleic acid delivery by magnetic force.

    Science.gov (United States)

    Plank, Christian; Anton, Martina; Rudolph, Carsten; Rosenecker, Joseph; Krötz, Florian

    2003-08-01

    Insufficient contact of inherently highly active nucleic acid delivery systems with target cells is a primary reason for their often observed limited efficacy. Physical methods of targeting can overcome this limitation and reduce the risk of undesired side effects due to non-target site delivery. The authors and others have developed a novel means of physical targeting, exploiting magnetic force acting on nucleic acid vectors associated with magnetic particles in order to mediate the rapid contact of vectors with target cells. Here, the principles of magnetic drug and nucleic acid delivery are reviewed, and the facts and potentials of the technique for research and therapeutic applications are discussed. Magnetically enhanced nucleic acid delivery - magnetofection - is universally applicable to viral and non-viral vectors, is extraordinarily rapid, simple and yields saturation level transfection at low dose in vitro. The method is useful for site-specific vector targeting in vivo. Exploiting the full potential of the technique requires an interdisciplinary research effort in magnetic field physics, magnetic particle chemistry, pharmaceutical formulation and medical application.

  10. A DNA origami nanorobot controlled by nucleic acid hybridization

    KAUST Repository

    Torelli, Emanuela

    2014-03-20

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

  11. Karyotype and nucleic acid content in Zantedeschia aethiopica Spr ...

    African Journals Online (AJOL)

    Analysis of karyotype, nucleic deoxyribonucleic acid (DNA) content and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were performed in Zantedeschia aethiopica and Zantedeschia elliottiana. Mitotic metaphase in both species showed 2n=32. The chromosomes of both species were quite similar ...

  12. Cell cycle nucleic acids, polypeptides and uses thereof

    Science.gov (United States)

    Gordon-Kamm, William J.; Lowe, Keith S.; Larkins, Brian A.; Dilkes, Brian R.; Sun, Yuejin

    2007-08-14

    The invention provides isolated nucleic acids and their encoded proteins that are involved in cell cycle regulation. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering cell cycle protein content, cell cycle progression, cell number and/or composition of plants.

  13. Predicting nucleic acid binding interfaces from structural models of proteins

    Science.gov (United States)

    Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

    2011-01-01

    The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared to patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. PMID:22086767

  14. Karyotype and nucleic acid content in Zantedeschia aethiopica Spr ...

    African Journals Online (AJOL)

    DR. NJ TONUKARI

    2012-07-03

    Jul 3, 2012 ... Analysis of karyotype, nucleic deoxyribonucleic acid (DNA) content and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were performed in Zantedeschia aethiopica and. Zantedeschia elliottiana. Mitotic metaphase in both species showed 2n=32. The chromosomes of both species ...

  15. Mosaic protein and nucleic acid vaccines against hepatitis C virus

    Science.gov (United States)

    Yusim, Karina; Korber, Bette T. M.; Kuiken, Carla L.; Fischer, William M.

    2013-06-11

    The invention relates to immunogenic compositions useful as HCV vaccines. Provided are HCV mosaic polypeptide and nucleic acid compositions which provide higher levels of T-cell epitope coverage while minimizing the occurrence of unnatural and rare epitopes compared to natural HCV polypeptides and consensus HCV sequences.

  16. Modulating gene function with peptide nucleic acids (PNA)

    DEFF Research Database (Denmark)

    Nielsen, Peter E.; Crooke, Stanley T.

    2008-01-01

    A review on peptide nucleic acid (PNA) oligomers as modulators of gene expression ranging from gene silencing at the mRNAor the dsDNA (antigene) level, and redirection of mRNA splicing to gene activation through transcription bubble mimicking. PNA chem., anti-infective agents, cellular delivery, ......, and in vivo bioavailability of PNA are briefly discussed. [on SciFinder (R)]...

  17. Assessment for Melting Temperature Measurement of Nucleic Acid by HRM

    Directory of Open Access Journals (Sweden)

    Jing Wang

    2016-01-01

    Full Text Available High resolution melting (HRM, with a high sensitivity to distinguish the nucleic acid species with small variations, has been widely applied in the mutation scanning, methylation analysis, and genotyping. For the aim of extending HRM for the evaluation of thermal stability of nucleic acid secondary structures on sequence dependence, we investigated effects of the dye of EvaGreen, metal ions, and impurities (such as dNTPs on melting temperature (Tm measurement by HRM. The accuracy of HRM was assessed as compared with UV melting method, and little difference between the two methods was found when the DNA Tm was higher than 40°C. Both insufficiency and excessiveness of EvaGreen were found to give rise to a little bit higher Tm, showing that the proportion of dye should be considered for precise Tm measurement of nucleic acids. Finally, HRM method was also successfully used to measure Tms of DNA triplex, hairpin, and RNA duplex. In conclusion, HRM can be applied in the evaluation of thermal stability of nucleic acid (DNA or RNA or secondary structural elements (even when dNTPs are present.

  18. A simple and rapid nucleic acid preparation method for reverse ...

    African Journals Online (AJOL)

    USER

    2010-03-29

    Mar 29, 2010 ... Key words: Nucleic acid preparation, reverse transcription polymerase chain reaction, dormant tubers, potato leafroll virus, potato virus S. INTRODUCTION. The vegetative propagation of potato (Solano tuberous. L.) presents ample opportunity for .... were amplified in 35 cycles. Annealing temperature was ...

  19. Non-enzymatic Polymerization of Nucleic Acids from Monomers

    DEFF Research Database (Denmark)

    Dörr, Mark; Löffler, Philipp M. G.; Monnard, Pierre-Alain

    2012-01-01

    This review deals with the state-of-the-art techniques in non-enzymatic nucleic acid condensation from monomers. In particular, the procedures called \\emph{monomer self-condensation} and \\emph{template-directed monomer condensation} are described, which have been developed to achieve efficient...

  20. Circulating nucleic acids as a diagnostic and prognostic marker in ...

    African Journals Online (AJOL)

    Elevated levels of circulating nucleic acids have been found in a variety of benign and malignant pathological conditions. The ability to detect and quantitate specific DNA, RNA and micro-RNA sequences in serum/ plasma has opened up the possibilities of non-invasive diagnosis and monitoring of diseases. Circulating ...

  1. PYRENE INTERCALATING NUCLEIC ACIDS WITH A CARBON LINKER

    DEFF Research Database (Denmark)

    Østergaard, Michael E.; Wamberg, Michael Chr.; Pedersen, Erik Bjerregaard

    2011-01-01

    geminally attached. Fluorescence studies of this intercalating nucleic acid with the pyrene moieties inserted as a bulge showed formation of an excimer band. When a mismatch was introduced at the site of the intercalator, an excimer band was formed for the destabilized duplexes whereas an exciplex band...

  2. Nucleic acid therapy for lifespan prolongation: Present and future

    Indian Academy of Sciences (India)

    Lifespan prolongation is a common desire of the human race. With advances in biotechnology, the mechanism of aging has been gradually unraveled, laying the theoretical basis of nucleic acid therapy for lifespan prolongation. Regretfully, clinically applicable interventions do not exist without the efforts of converting theory ...

  3. Prediction of molecular alignment of nucleic acids in aligned media

    NARCIS (Netherlands)

    Wu, B.; Petersen, M.; Girard, F.C.; Tessari, M.; Wijmenga, S.S.

    2006-01-01

    We demonstrate - using the data base of all deposited DNA and RNA structures aligned in Pf1-medium and RDC refined - that for nucleic acids in a Pf1-medium the electrostatic alignment tensor can be predicted reliably and accurately via a simple and fast calculation based on the gyration tensor

  4. Predicting nucleic acid binding interfaces from structural models of proteins.

    Science.gov (United States)

    Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

    2012-02-01

    The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.

  5. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2017-09-05

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  6. Surface plasmon resonance sensing of nucleic acids: A review

    Czech Academy of Sciences Publication Activity Database

    Šípová, Hana; Homola, Jiří

    -, č. 773 (2013), s. 9-23 ISSN 0003-2670 R&D Projects: GA MŠk(CZ) LH11102 Institutional support: RVO:67985882 Keywords : Surface plasmon resonance * Nucleic acid * Biosensor Subject RIV: JB - Sensors, Measurment, Regulation Impact factor: 4.517, year: 2013

  7. Nucleic acid detection with surface plasmon resonance using cationic latex

    NARCIS (Netherlands)

    de Vries, E.F.A.; Schasfoort, Richardus B.M.; van der Plas, J.; Greve, Jan

    1994-01-01

    An affinity sensor based on Surface Plasmon Resonance (SPR) was used to detect nucleic acids. SPR is an optical technique that is able to detect small changes in the refractive index of the immediate vicinity of a metal surface. After a specific amplification of DNA, achieved using the polymerase

  8. nucleic acid amplification as used in the diagnosis and management ...

    African Journals Online (AJOL)

    DR. AMINU

    2014-06-01

    Jun 1, 2014 ... thus, amplification based methods offer superior performance ... self-sustaining sequence replication), Amplification of nucleic acid probe (e.g., ligase chain reaction and Q-beta replicase) and Signal amplification (e.g., branched-probe DNA assay). PCR ... advantages, limitations and clinical utility (Fredricks.

  9. Watson-Crick hydrogen bonding of unlocked nucleic acids

    DEFF Research Database (Denmark)

    Langkjær, Niels; Wengel, Jesper; Pasternak, Anna

    2015-01-01

    We herein describe the synthesis of two new unlocked nucleic acid building blocks containing hypoxanthine and 2,6-diaminopurine as nucleobase moieties and their incorporation into oligonucleotides. The modified oligonucleotides were used to examine the thermodynamic properties of UNA against unmo...... unmodified oligonucleotides and the resulting thermodynamic data support that the hydrogen bonding face of UNA is Watson-Crick like....

  10. A nucleic acid dependent chemical photocatalysis in live human cells

    DEFF Research Database (Denmark)

    Arian, Dumitru; Cló, Emiliano; Gothelf, Kurt V

    2010-01-01

    Only two nucleic acid directed chemical reactions that are compatible with live cells have been reported to date. Neither of these processes generate toxic species from nontoxic starting materials. Reactions of the latter type could be applied as gene-specific drugs, for example, in the treatment...

  11. The use of solid supports to generate nucleic acid carriers.

    Science.gov (United States)

    Unciti-Broceta, Asier; Díaz-Mochón, Juan José; Sánchez-Martín, Rosario M; Bradley, Mark

    2012-07-17

    Nucleic acids are the foundation stone of all cellular processes. Consequently, the use of DNA or RNA to treat genetic and acquired disorders (so called gene therapy) offers enormous potential benefits. The restitution of defective genes or the suppression of malignant genes could target a range of diseases, including cancers, inherited diseases (cystic fibrosis, muscular dystrophy, etc.), and viral infections. However, this strategy has a major barrier: the size and charge of nucleic acids largely restricts their transit into eukaryotic cells. Potential strategies to solve this problem include the use of a variety of natural and synthetic nucleic acid carriers. Driven by the aim and ambition of translating this promising therapeutic approach into the clinic, researchers have been actively developing advanced delivery systems for nucleic acids for more than 20 years. A decade ago we began our investigations of solid-phase techniques to construct families of novel nucleic acid carriers for transfection. We envisaged that the solid-phase synthesis of polycationic dendrimers and derivatized polyamimes would offer distinct advantages over solution phase techniques. Notably in solid phase synthesis we could take advantage of mass action and streamlined purification procedures, while simplifying the handling of compounds with high polarities and plurality of functional groups. Parallel synthesis methods would also allow rapid access to libraries of compounds with improved purities and yields over comparable solution methodologies and facilitate the development of structure activity relationships. We also twisted the concept of the solid-phase support on its head: we devised miniaturized solid supports that provided an innovative cell delivery vehicle in their own right, carrying covalently conjugated cargos (biomolecules) into cells. In this Account, we summarize the main outcomes of this series of chemically related projects.

  12. Modeling nucleic acid structure in the presence of single-stranded binding proteins

    Science.gov (United States)

    Forties, Robert; Bundschuh, Ralf

    2009-03-01

    There are many important proteins which bind single-stranded nucleic acids, such as the nucleocapsid protein in HIV, the RecA DNA repair protein in bacteria, and all proteins involved in mRNA splicing and translation. We extend the Vienna Package for quantitatively modeling the secondary structure of nucleic acids to include proteins which bind to unpaired portions of the nucleic acid. All parameters needed to model nucleic acid secondary structures in the absence of proteins have been previously measured. This leaves the footprint and sequence dependent binding affinity of the protein as adjustable parameters of our model. Using this model we are able to predict the probability of the protein binding at any position in the nucleic acid sequence, the impact of the protein on nucleic acid base pairing, the end-to-end distance distribution for the nucleic acid, and FRET distributions for fluorophores attached to the nucleic acid.

  13. Polymerase chain reaction system using magnetic beads for analyzing a sample that includes nucleic acid

    Science.gov (United States)

    Nasarabadi, Shanavaz [Livermore, CA

    2011-01-11

    A polymerase chain reaction system for analyzing a sample containing nucleic acid includes providing magnetic beads; providing a flow channel having a polymerase chain reaction chamber, a pre polymerase chain reaction magnet position adjacent the polymerase chain reaction chamber, and a post pre polymerase magnet position adjacent the polymerase chain reaction chamber. The nucleic acid is bound to the magnetic beads. The magnetic beads with the nucleic acid flow to the pre polymerase chain reaction magnet position in the flow channel. The magnetic beads and the nucleic acid are washed with ethanol. The nucleic acid in the polymerase chain reaction chamber is amplified. The magnetic beads and the nucleic acid are separated into a waste stream containing the magnetic beads and a post polymerase chain reaction mix containing the nucleic acid. The reaction mix containing the nucleic acid flows to an analysis unit in the channel for analysis.

  14. 78 FR 36698 - Microbiology Devices; Reclassification of Nucleic Acid-Based Systems for Mycobacterium tuberculosis

    Science.gov (United States)

    2013-06-19

    ... Nucleic Acid-Based Systems for Mycobacterium tuberculosis Complex in Respiratory Specimens AGENCY: Food...) is proposing to reclassify nucleic acid-based in vitro diagnostic devices for the detection of... Controls Guideline: Nucleic Acid-Based In Vitro Diagnostic Devices for the Detection of Mycobacterium...

  15. Nucleic acid-based fluorescent probes and their analytical potential.

    Science.gov (United States)

    Juskowiak, Bernard

    2011-03-01

    It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays.

  16. Fool's Gold Footprinting: microfluidic probing of nucleic acids

    Science.gov (United States)

    Jones, Christopher D.; Schlatterer, Joerg C.; Brenowitz, Michael; Pollack, Lois

    2012-02-01

    We describe a microfluidic device containing a mineral matrix capable of rapidly generating hydroxyl radicals that enables high-resolution structural studies of nucleic acids. Hydroxyl radicals cleave the solvent accessible backbone of DNA and RNA; the cleavage products can be detected with as fine as single nucleotide resolution. Protection from hydroxyl radical cleavage (footprinting) can identify sites of protein binding or the presence of tertiary structure. Here we report preparation of micron sized particles of iron sulfide (pyrite) and fabrication of a microfluidic prototype that together generate enough hydroxyl radicals within 20 ms to cleave DNA sufficiently for a footprinting analysis to be conducted. This prototype enables the development of high-throughput and/or rapid reaction devices with which to probe nucleic acid folding dynamics and ligand binding.

  17. NPIDB: Nucleic acid-Protein Interaction DataBase.

    Science.gov (United States)

    Kirsanov, Dmitry D; Zanegina, Olga N; Aksianov, Evgeniy A; Spirin, Sergei A; Karyagina, Anna S; Alexeevski, Andrei V

    2013-01-01

    The Nucleic acid-Protein Interaction DataBase (http://npidb.belozersky.msu.ru/) contains information derived from structures of DNA-protein and RNA-protein complexes extracted from the Protein Data Bank (3846 complexes in October 2012). It provides a web interface and a set of tools for extracting biologically meaningful characteristics of nucleoprotein complexes. The content of the database is updated weekly. The current version of the Nucleic acid-Protein Interaction DataBase is an upgrade of the version published in 2007. The improvements include a new web interface, new tools for calculation of intermolecular interactions, a classification of SCOP families that contains DNA-binding protein domains and data on conserved water molecules on the DNA-protein interface.

  18. Devices, systems, and methods for detecting nucleic acids using sedimentation

    Energy Technology Data Exchange (ETDEWEB)

    Koh, Chung-Yan; Schaff, Ulrich Y.; Sommer, Gregory J.

    2017-10-24

    Embodiments of the present invention are directed toward devices, systems, and method for conducting nucleic acid purification and quantification using sedimentation. In one example, a method includes generating complexes which bind to a plurality of beads in a fluid sample, individual ones of the complexes comprising a nucleic acid molecule such as DNA or RNA and a labeling agent. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a density lower than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

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

    Science.gov (United States)

    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.

  20. Surface plasmon resonance sensing of nucleic acids: A review

    Energy Technology Data Exchange (ETDEWEB)

    Šípová, Hana [Institute of Photonics and Electronics, Academy of Sciences of the Czech Republic, Chaberská 57, Prague (Czech Republic); Homola, Jiří, E-mail: homola@ufe.cz [Institute of Photonics and Electronics, Academy of Sciences of the Czech Republic, Chaberská 57, Prague (Czech Republic)

    2013-04-22

    Highlights: ► Advances of nucleic acid (NA) surface plasmon resonance (SPR) sensors are presented. ► Bioanalytical applications of NA SPR biosensors are reviewed. ► Applications for study of molecular interactions involving NAs are also discussed. -- Abstract: Biosensors based on surface plasmon resonance (SPR) have become a central tool for the investigation and quantification of biomolecules and their interactions. Nucleic acids (NAs) play a vital role in numerous biological processes and therefore have been one of the major groups of biomolecules targeted by the SPR biosensors. This paper discusses the advances of NA SPR biosensor technology and reviews its applications both in the research of molecular interactions involving NAs (NA–NA, NA–protein, NA–small molecule), as well as for the field of bioanalytics in the areas of food safety, medical diagnosis and environmental monitoring.

  1. An extended IUPAC nomenclature code for polymorphic nucleic acids.

    Science.gov (United States)

    Johnson, Andrew D

    2010-05-15

    The International Union of Pure and Applied Chemistry (IUPAC) code specified nearly 25 years ago provides a nomenclature for incompletely specified nucleic acids. However, no system currently exists that allows for the informatics representation of the relative abundance at polymorphic nucleic acids (e.g. single nucleotide polymorphisms) in a single specified character, or a string of characters. Here, I propose such an information code as a natural extension to the IUPAC nomenclature code, and present some potential uses and limitations to such a code. The primary anticipated use of this extended nomenclature code is to assist in the representation of the rapidly growing space of information in human genetic variation. johnsonad2@nhlbi.nih.gov Supplementary data are available at Bioinformatics online.

  2. Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support

    Directory of Open Access Journals (Sweden)

    Leclerc Mario

    2005-04-01

    Full Text Available Abstract Background Nucleic acids detection using microarrays requires labelling of target nucleic acids with fluorophores or other reporter molecules prior to hybridization. Results Using surface-bound peptide nucleic acids (PNA probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray. Conclusion This simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries. This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes.

  3. Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support

    Science.gov (United States)

    Raymond, Frédéric R; Ho, Hoang-Anh; Peytavi, Régis; Bissonnette, Luc; Boissinot, Maurice; Picard, François J; Leclerc, Mario; Bergeron, Michel G

    2005-01-01

    Background Nucleic acids detection using microarrays requires labelling of target nucleic acids with fluorophores or other reporter molecules prior to hybridization. Results Using surface-bound peptide nucleic acids (PNA) probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray. Conclusion This simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries. This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes. PMID:15850478

  4. Present status of protein and nucleic acid database activities in the world

    Science.gov (United States)

    Tsugita, Akira

    The first protein database was founded in 1965, followed by the establishment of nucleic acid databases from 1971. Presently there are six major sequence databases, located in Japan, USA and the FRG-three for protein data and three for nucleic acid data. International cooperation between the protein databases and between the nucleic acid databases have greatly facilitated compilation and dissemination of data. Coordination between these protein and nucleic acid databases have progressed with the support of the CODATA Task Group and the International Advisory Board for Nucleic Acid Databases. In the protein field, several additional database activities are initiated to contribute to protein engineering and structure-activity relationships.

  5. Silicon Dioxide Thin Film Mediated Single Cell Nucleic Acid Isolation

    Science.gov (United States)

    Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

    2013-01-01

    A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube. PMID:23874571

  6. Developing nucleic acid-based electrical detection systems.

    Science.gov (United States)

    Gabig-Ciminska, Magdalena

    2006-03-02

    Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical

  7. Development of Sorbents for Extraction and Stabilization of Nucleic Acids

    Science.gov (United States)

    2016-09-13

    biomolecules, espe - cially DNA and RNA. The goal was to provide stabilization methods for reagents and targets in order to allow for a wider range of...within the scaffolds, and optimal methodologies for using the systems to capture, stabilize, and recover nucleic acids. Sorbent design...currently in use. In addition, stabilizing technologies can provide new sampling methodologies that can enhance the capacity for obtaining environmental

  8. Watson-Crick hydrogen bonding of unlocked nucleic acids.

    Science.gov (United States)

    Langkjær, Niels; Wengel, Jesper; Pasternak, Anna

    2015-11-15

    We herein describe the synthesis of two new unlocked nucleic acid building blocks containing hypoxanthine and 2,6-diaminopurine as nucleobase moieties and their incorporation into oligonucleotides. The modified oligonucleotides were used to examine the thermodynamic properties of UNA against unmodified oligonucleotides and the resulting thermodynamic data support that the hydrogen bonding face of UNA is Watson-Crick like. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Los Alamos sequence analysis package for nucleic acids and proteins.

    OpenAIRE

    Kanehisa, M I

    1982-01-01

    An interactive system for computer analysis of nucleic acid and protein sequences has been developed for the Los Alamos DNA Sequence Database. It provides a convenient way to search or verify various sequence features, e.g., restriction enzyme sites, protein coding frames, and properties of coded proteins. Further, the comprehensive analysis package on a large-scale database can be used for comparative studies on sequence and structural homologies in order to find unnoted information stored i...

  10. Developing nucleic acid-based electrical detection systems

    Directory of Open Access Journals (Sweden)

    Gabig-Ciminska Magdalena

    2006-03-01

    Full Text Available Abstract Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in

  11. Developing nucleic acid-based electrical detection systems

    Science.gov (United States)

    Gabig-Ciminska, Magdalena

    2006-01-01

    Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical

  12. Molecular modeling of nucleic Acid structure: electrostatics and solvation.

    Science.gov (United States)

    Bergonzo, Christina; Galindo-Murillo, Rodrigo; Cheatham, Thomas E

    2014-12-19

    This unit presents an overview of computer simulation techniques as applied to nucleic acid systems, ranging from simple in vacuo molecular modeling techniques to more complete all-atom molecular dynamics treatments that include an explicit representation of the environment. The third in a series of four units, this unit focuses on critical issues in solvation and the treatment of electrostatics. UNITS 7.5 & 7.8 introduced the modeling of nucleic acid structure at the molecular level. This included a discussion of how to generate an initial model, how to evaluate the utility or reliability of a given model, and ultimately how to manipulate this model to better understand its structure, dynamics, and interactions. Subject to an appropriate representation of the energy, such as a specifically parameterized empirical force field, the techniques of minimization and Monte Carlo simulation, as well as molecular dynamics (MD) methods, were introduced as a way of sampling conformational space for a better understanding of the relevance of a given model. This discussion highlighted the major limitations with modeling in general. When sampling conformational space effectively, difficult issues are encountered, such as multiple minima or conformational sampling problems, and accurately representing the underlying energy of interaction. In order to provide a realistic model of the underlying energetics for nucleic acids in their native environments, it is crucial to include some representation of solvation (by water) and also to properly treat the electrostatic interactions. These subjects are discussed in detail in this unit. Copyright © 2014 John Wiley & Sons, Inc.

  13. Universal nucleic acids sample preparation method for cells, spores and their mixture

    Science.gov (United States)

    Bavykin, Sergei [Darien, IL

    2011-01-18

    The present invention relates to a method for extracting nucleic acids from biological samples. More specifically the invention relates to a universal method for extracting nucleic acids from unidentified biological samples. An advantage of the presently invented method is its ability to effectively and efficiently extract nucleic acids from a variety of different cell types including but not limited to prokaryotic or eukaryotic cells and/or recalcitrant organisms (i.e. spores). Unlike prior art methods which are focused on extracting nucleic acids from vegetative cell or spores, the present invention effectively extracts nucleic acids from spores, multiple cell types or mixtures thereof using a single method. Important that the invented method has demonstrated an ability to extract nucleic acids from spores and vegetative bacterial cells with similar levels effectiveness. The invented method employs a multi-step protocol which erodes the cell structure of the biological sample, isolates, labels, fragments nucleic acids and purifies labeled samples from the excess of dye.

  14. Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges.

    Science.gov (United States)

    Qing, Taiping; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Wen, Li; Shangguan, Jingfang; Mao, Zhengui; Lei, Yanli

    2016-04-01

    Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as 'nano-tools' for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors.

  15. Origin of Overstretching Transitions in Single-Stranded Nucleic Acids

    Science.gov (United States)

    Scholl, Zackary N.; Rabbi, Mahir; Lee, David; Manson, Laura; S-Gracz, Hanna; Marszalek, Piotr E.

    2013-11-01

    We combined single-molecule force spectroscopy with nuclear magnetic resonance measurements and molecular mechanics simulations to examine overstretching transitions in single-stranded nucleic acids. In single-stranded DNA and single-stranded RNA there is a low-force transition that involves unwinding of the helical structure, along with base unstacking. We determined that the high-force transition that occurs in polydeoxyadenylic acid single-stranded DNA is caused by the cooperative forced flipping of the dihedral angle formed between four atoms, O5’-C5’-C4’-C3’ (γ torsion), in the nucleic acid backbone within the canonical B-type helix. The γ torsion also flips under force in A-type helices, where the helix is shorter and wider as compared to the B-type helix, but this transition is less cooperative than in the B type and does not generate a high-force plateau in the force spectrums of A-type helices. We find that a similar high-force transition can be induced in polyadenylic acid single-stranded RNA by urea, presumably due to disrupting the intramolecular hydrogen bonding in the backbone. We hypothesize that a pronounced high-force transition observed for B-type helices of double stranded DNA also involves a cooperative flip of the γ torsion. These observations suggest new fundamental relationships between the canonical structures of single-and double-stranded DNA and the mechanism of their molecular elasticity.

  16. Highly simplified lateral flow-based nucleic acid sample preparation and passive fluid flow control

    Science.gov (United States)

    Cary, Robert E.

    2015-12-08

    Highly simplified lateral flow chromatographic nucleic acid sample preparation methods, devices, and integrated systems are provided for the efficient concentration of trace samples and the removal of nucleic acid amplification inhibitors. Methods for capturing and reducing inhibitors of nucleic acid amplification reactions, such as humic acid, using polyvinylpyrrolidone treated elements of the lateral flow device are also provided. Further provided are passive fluid control methods and systems for use in lateral flow assays.

  17. Assays for urinary biomarkers of oxidatively damaged nucleic acids

    DEFF Research Database (Denmark)

    Weimann, Allan; Broedbaek, Kasper; Henriksen, Trine

    2012-01-01

    -linked immunosorbent assay). The major analytical challenge is specificity. The best combination of selectivity and speed of analysis can be obtained by liquid chromatography coupled with tandem mass spectrometric detection. This, however, is also the most demanding technique with regard to price, complexity...... and skills requirement. The available ELISA methods present considerable specificity problems and cannot be recommended at present. The oxidized nucleic acid metabolites in urine are assumed to originate from the DNA and RNA. However, direct evidence is not available. A possible contribution from...

  18. Advances in nucleic acid-based diagnostics of bacterial infections

    DEFF Research Database (Denmark)

    Barken, Kim Bundvig; Haagensen, Janus Anders Juul; Tolker-Nielsen, Tim

    2007-01-01

    Methods for rapid detection of infectious bacteria and antimicrobial-resistant pathogens have evolved significantly over the last decade. Many of the new procedures are nucleic acid-based and replace conventional diagnostic methods like culturing which is time consuming especially with fastidious...... of these pathogens is important to isolate patients and prevent further spreading of the diseases. Newly developed diagnostic procedures are superior with respect to turnaround time, sensitivity and specificity. Methods like multiplex real time PCR and different array-based technologies offer the possibility...

  19. Efficient liposome fusion mediated by lipid-nucleic acid conjugates

    DEFF Research Database (Denmark)

    Ries, O; Löffler, P M G; Rabe, A

    2017-01-01

    The fusion of biomembranes with release of encapsulated content in a controlled way is crucial for cell signaling, endo- and exocytosis and intracellular trafficking. Programmable fusion of liposomes and an efficient mixing of their contents have the potential to enable the study of chemical...... and enzymatic processes in a confined environment and under crowded conditions outside biological systems. We report on DNA-controlled fusion of lipid bilayer membranes using lipid-nucleic acid conjugates (LiNAs) to mediate lipid and content mixing of liposomes. Screening of different membrane anchor and linker...

  20. Nucleic acid constructs containing orthogonal site selective recombinases (OSSRs)

    Energy Technology Data Exchange (ETDEWEB)

    Gilmore, Joshua M.; Anderson, J. Christopher; Dueber, John E.

    2017-08-29

    The present invention provides for a recombinant nucleic acid comprising a nucleotide sequence comprising a plurality of constructs, wherein each construct independently comprises a nucleotide sequence of interest flanked by a pair of recombinase recognition sequences. Each pair of recombinase recognition sequences is recognized by a distinct recombinase. Optionally, each construct can, independently, further comprise one or more genes encoding a recombinase capable of recognizing the pair of recombinase recognition sequences of the construct. The recombinase can be an orthogonal (non-cross reacting), site-selective recombinase (OSSR).

  1. Studies on Nucleic Acids – Structure and Dynamics

    OpenAIRE

    Isaksson, Johan

    2005-01-01

    This thesis is based on six papers, Papers I-VI, focusing on the interplay between the stabilizing elements of nucleic acids self-assembly; hydrogen bonding, stacking and solvent effects. In Paper I we investigate how the substitution of the O4' for CH2 in the sugar moiety of adenosine (2'-deoxyaristeromycin) at the A6 position of the Dickerson-Drew dodecamer makes the two modified bases exist in a dynamic equilibrium between Hoogsteen and Watson-Crick base pairing in the NMR time scale. Pape...

  2. Design Considerations for RNA Spherical Nucleic Acids (SNAs).

    Science.gov (United States)

    Barnaby, Stacey N; Perelman, Grant A; Kohlstedt, Kevin L; Chinen, Alyssa B; Schatz, George C; Mirkin, Chad A

    2016-09-21

    Ribonucleic acids (RNAs) are key components in many cellular processes such as cell division, differentiation, growth, aging, and death. RNA spherical nucleic acids (RNA-SNAs), which consist of dense shells of double-stranded RNA on nanoparticle surfaces, are powerful and promising therapeutic modalities because they confer advantages over linear RNA such as high cellular uptake and enhanced stability. Due to their three-dimensional shell of oligonucleotides, SNAs, in comparison to linear nucleic acids, interact with the biological environment in unique ways. Herein, the modularity of the RNA-SNA is used to systematically study structure-function relationships in order to understand how the oligonucleotide shell affects interactions with a specific type of biological environment, namely, one that contains serum nucleases. We use a combination of experiment and theory to determine the key architectural properties (i.e., sequence, density, spacer moiety, and backfill molecule) that affect how RNA-SNAs interact with serum nucleases. These data establish a set of design parameters for SNA architectures that are optimized in terms of stability.

  3. Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes.

    Science.gov (United States)

    Marras, Salvatore A E; Tyagi, Sanjay; Kramer, Fred Russell

    2006-01-01

    A number of formats for nucleic acid hybridization have been developed to identify DNA and RNA sequences that are involved in cellular processes and that aid in the diagnosis of genetic and infectious diseases. The introduction of hybridization probes with interactive fluorophore pairs has enabled the development of homogeneous hybridization assays for the direct identification of nucleic acids. A change in the fluorescence of these probes indicates the presence of a target nucleic acid, and there is no need to separate unbound probes from hybridized probes. The advantages of homogeneous hybridization assays are their speed and simplicity. In addition, homogeneous assays can be combined with nucleic acid amplification, enabling the detection of rare target nucleic acids. These assays can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations.

  4. Introduction of structural affinity handles as a tool in selective nucleic acid separations

    Science.gov (United States)

    Willson, III, Richard Coale (Inventor); Cano, Luis Antonio (Inventor)

    2011-01-01

    The method is used for separating nucleic acids and other similar constructs. It involves selective introduction, enhancement, or stabilization of affinity handles such as single-strandedness in the undesired (or desired) nucleic acids as compared to the usual structure (e.g., double-strandedness) of the desired (or undesired) nucleic acids. The undesired (or desired) nucleic acids are separated from the desired (or undesired) nucleic acids due to capture by methods including but not limited to immobilized metal affinity chromatography, immobilized single-stranded DNA binding (SSB) protein, and immobilized oligonucleotides. The invention is useful to: remove contaminating genomic DNA from plasmid DNA; remove genomic DNA from plasmids, BACs, and similar constructs; selectively separate oligonucleotides and similar DNA fragments from their partner strands; purification of aptamers, (deoxy)-ribozymes and other highly structured nucleic acids; Separation of restriction fragments without using agarose gels; manufacture recombinant Taq polymerase or similar products that are sensitive to host genomic DNA contamination; and other applications.

  5. Templated Synthesis of Peptide Nucleic Acids via Sequence-Selective Base-Filling Reactions

    OpenAIRE

    Heemstra, Jennifer M.; Liu, David R.

    2009-01-01

    The templated synthesis of nucleic acids has previously been achieved through the backbone ligation of preformed nucleotide monomers or oligomers. In contrast, here we demonstrate templated nucleic acid synthesis using a base-filling approach in which individual bases are added to abasic sites of a peptide nucleic acid (PNA). Because nucleobase substrates in this approach are not self-reactive, a base-filling approach may reduce the formation of nontemplated reaction products. Using either re...

  6. Easily denaturing nucleic acids derived from intercalating nucleic acids: thermal stability studies, dual duplex invasion and inhibition of transcription start

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V; Vester, Birte; Hansen, Lykke Haastrup

    2005-01-01

    The bulged insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (monomer P) in two complementary 8mer DNA strands (intercalating nucleic acids) opposite to each other resulted in the formation of an easily denaturing duplex, which had lower thermal stability (21.0 degrees C) than the wild-type double......-stranded DNA (dsDNA, 26.0 degrees C), but both modified oligodeoxynucleotides had increased binding affinity toward complementary single-stranded DNA (ssDNA) (41.5 and 39.0 degrees C). Zipping of pyrene moieties in an easily denaturing duplex gave formation of a strong excimer band at 480 nm upon excitation...

  7. Constrained Multistate Sequence Design for Nucleic Acid Reaction Pathway Engineering.

    Science.gov (United States)

    Wolfe, Brian R; Porubsky, Nicholas J; Zadeh, Joseph N; Dirks, Robert M; Pierce, Niles A

    2017-03-01

    We describe a framework for designing the sequences of multiple nucleic acid strands intended to hybridize in solution via a prescribed reaction pathway. Sequence design is formulated as a multistate optimization problem using a set of target test tubes to represent reactant, intermediate, and product states of the system, as well as to model crosstalk between components. Each target test tube contains a set of desired "on-target" complexes, each with a target secondary structure and target concentration, and a set of undesired "off-target" complexes, each with vanishing target concentration. Optimization of the equilibrium ensemble properties of the target test tubes implements both a positive design paradigm, explicitly designing for on-pathway elementary steps, and a negative design paradigm, explicitly designing against off-pathway crosstalk. Sequence design is performed subject to diverse user-specified sequence constraints including composition constraints, complementarity constraints, pattern prevention constraints, and biological constraints. Constrained multistate sequence design facilitates nucleic acid reaction pathway engineering for diverse applications in molecular programming and synthetic biology. Design jobs can be run online via the NUPACK web application.

  8. Nucleic Acid Drugs for Prevention of Cardiac Rejection

    Directory of Open Access Journals (Sweden)

    Jun-ichi Suzuki

    2009-01-01

    Full Text Available Heart transplantation has been broadly performed in humans. However, occurrence of acute and chronic rejection has not yet been resolved. Several inflammatory factors, such as cytokines and adhesion molecules, enhance the rejection. The graft arterial disease (GAD, which is a type of chronic rejection, is characterized by intimal thickening comprised of proliferative smooth muscle cells. Specific treatments that target the attenuation of acute rejection and GAD formation have not been well studied in cardiac transplantation. Recent progress in the nucleic acid drugs, such as antisense oligodeoxynucleotides (ODNs to regulate the transcription of disease-related genes, has important roles in therapeutic applications. Transfection of cis-element double-stranded DNA, named as “decoy,” has been also reported to be a useful nucleic acid drug. This decoy strategy has been not only a useful method for the experimental studies of gene regulation but also a novel clinical strategy. In this paper, we reviewed the experimental results of NF-κB, E2F, AP-1, and STAT-1 decoy and other ODNs using the experimental heart transplant models.

  9. Functional nucleic acids as in vivo metabolite and ion biosensors.

    Science.gov (United States)

    Alsaafin, Alaa; McKeague, Maureen

    2017-08-15

    Characterizing the role of metabolites, metals, and proteins is required to understand normal cell function, and ultimately, elucidate the mechanism of disease. Metabolite concentration and transformation results collected from cell lysates or fixed-cells conceal important dynamic information and differences between individual cells that often have profound functional consequences. Functional nucleic acid-based biosensors are emerging tools that are capable of monitoring ions and metabolites in cell populations or whole animals. Functional nucleic acids (FNAs) are a class of biomolecules that can exhibit either ligand binding or enzymatic activity. Unlike their protein analogues or the use of instrument-based analysis, FNA-based biosensors are capable of entering cells without disruption to the cellular environment and can report on the concentration, dynamics, and spatial localization of molecules in cells. Here, we review the types of FNAs that have been used as in vivo biosensors, and how FNAs can be coupled to transduction systems and delivered inside cells. We also provide examples from the literature that demonstrate their impact in practical applications. Finally, we comment on the critical limitations that need to be addressed to enable their use for single-cell dynamic tracking of metabolites and ions in vivo. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  10. Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology.

    Science.gov (United States)

    Chambers, Cecilia R; Patrick, Wayne M

    2015-01-01

    With their ability to catalyse the formation of phosphodiester linkages, DNA ligases and RNA ligases are essential tools for many protocols in molecular biology and biotechnology. Currently, the nucleic acid ligases from bacteriophage T4 are used extensively in these protocols. In this review, we argue that the nucleic acid ligases from Archaea represent a largely untapped pool of enzymes with diverse and potentially favourable properties for new and emerging biotechnological applications. We summarise the current state of knowledge on archaeal DNA and RNA ligases, which makes apparent the relative scarcity of information on in vitro activities that are of most relevance to biotechnologists (such as the ability to join blunt- or cohesive-ended, double-stranded DNA fragments). We highlight the existing biotechnological applications of archaeal DNA ligases and RNA ligases. Finally, we draw attention to recent experiments in which protein engineering was used to modify the activities of the DNA ligase from Pyrococcus furiosus and the RNA ligase from Methanothermobacter thermautotrophicus, thus demonstrating the potential for further work in this area.

  11. Characterization of a crosslinked nucleic acid - helix destabilizing protein complex

    Energy Technology Data Exchange (ETDEWEB)

    Karpel, R.L.; Levin, V.Y.; Haley, B.E.

    1986-05-01

    They have enzymatically synthesized /sup 3/H- and /sup 32/P-poly(A,8N/sub 3/A) from 8-N/sub 3/ADP and radiolabeled ADP, and have used this polynucleotide to photoaffinity label T4 gene 32 protein, as well as several other helix-destabilizing proteins (HDPs). Irradiation of /sup 32/P-/sup 3/H-poly(A,N/sub 3/A) mixtures for short durations produces a covalent complex, seen as a high molecular weight, radioactive band on SDS-polyacrylamide gels. Preliminary experiments on other HDPs, from prokaryotic, eukaryotic and animal viral sources, show analogous results. Several successful control experiments indicate that this system is suitable for binding site localization on /sup 32/P. Single-stranded nucleic acids competitively inhibit photolabeling. The effect of NaCl on photolabeling parallels the salt-dependence of /sup 32/P-poly(A,N/sub 3/A) binding. Photolabeling reaches a plateau after approx.1 min, and the formation of the high molecular weight complex parallels the reduction of free /sup 32/P on SDS gels. Staph. nuclease digestion of crosslinked complexes produces a diffuse, radioactive band on SDS gels, migrating just behind free /sup 32/P. When these digested complexes are subjected to reverse-phase HPLC on a C3 Ultrapore column, the nucleic acid /sup 32/P-label is seen to coelute with protein. They are currently employing RP-HPLC methods to locate the label on tryptic peptides of nuclease-digested complexes.

  12. Nucleic acid delivery: the missing pieces of the puzzle?

    Science.gov (United States)

    Nguyen, Juliane; Szoka, Francis C

    2012-07-17

    The delivery of genes or RNA interference (RNAi) agents can increase or decrease the expression of virtually any protein in a cell, and this process opens the path for cures to most diseases that afflict humans. However, the high molecular weight, anionic nature, and instability of nucleic acids in the presence of enzymes pose major obstacles to their delivery and frustrates their use as human therapies. This Account describes current ideas about the mechanisms in nonviral nucleic acid delivery and how lipidic and polymeric carriers can overcome some of the critical barriers to delivery. Over the last 20 years, researchers have developed a multitude of polymeric and lipidic vectors, but only a small fraction of these have progressed into clinical trials. None of these vectors has received FDA approval, which indicates that the current vectors do not yet have suitable properties for effective in vivo nucleic acid delivery. Nucleic acid delivery is a multistep process and inefficiencies at any stage result in a dramatic decrease in gene delivery or gene silencing. However, the majority of studies investigating synthetic vectors focus solely on optimization of endosomal escape. A small number of studies address how to improve uptake via targeted delivery, and an even smaller fraction examine the intracellular fate of the delivery systems and nucleic acid cargo. The internalization of genes into the cell nucleus remains an inefficient and mysterious process. In the case of DNA delivery, strategies are needed to increase and accelerate the migration of DNA through the cytoplasm and transport it through the nuclear membrane. siRNA delivery involves fewer barriers. siRNA is more readily released from the carrier and more resistant to enzymatic degradation, and its target is in the cytoplasm; hence, siRNA delivery systems are becoming a clinical reality. With regard to siRNA therapy, the exact cytoplasmic location of RNA-induced silencing complex (RISC) formation and

  13. Versatile phosphoramidation reactions for nucleic acid conjugations with peptides, proteins, chromophores, and biotin derivatives.

    Science.gov (United States)

    Wang, Tzu-Pin; Chiou, Yi-Jang; Chen, Yi; Wang, Eng-Chi; Hwang, Long-Chih; Chen, Bing-Hung; Chen, Yen-Hsu; Ko, Chun-Han

    2010-09-15

    Chemical conjugations of nucleic acids with macromolecules or small molecules are common approaches to study nucleic acids in chemistry and biology and to exploit nucleic acids for medical applications. The conjugation of nucleic acids such as oligonucleotides with peptides is especially useful to circumvent cell delivery and specificity problems of oligonucleotides as therapeutic agents. However, current approaches are limited and inefficient in their ability to afford peptide-oligonucleotide conjugates (POCs). Here, we report an effective and reproducible approach to prepare POCs and other nucleic acid conjugates based on a newly developed nucleic acid phosphoramidation method. The development of a new nucleic acid phosphoramidation reaction was achieved by our successful synthesis of a novel amine-containing biotin derivative used to systematically optimize the reactions. The improved phosphoramidation reactions dramatically increased yields of nucleic acid-biotin conjugates up to 80% after 3 h reaction. Any nucleic acids with a terminal phosphate group are suitable reactants in phosphoramidation reactions to conjugate with amine-containing molecules such as biotin and fluorescein derivatives, proteins, and, most importantly, peptides to enable the synthesis of POCs for therapeutic applications. Polymerase chain reactions (PCRs) to study incorporation of biotin or fluorescein-tagged DNA primers into the reaction products demonstrated that appropriate controls of nucleic acid phosphoramidation reactions incur minimum adverse effects on inherited base-pairing characteristics of nucleotides in nucleic acids. The phosphoramidation approach preserves the integrity of hybridization specificity in nucleic acids when preparing POCs. By retaining integrity of the nucleic acids, their effectiveness as therapeutic reagents for gene silencing, gene therapy, and RNA interference is ensured. The potential for POC use was demonstrated by two-step phosphoramidation reactions to

  14. Nucleic acid sample preparation for in vitro molecular diagnosis: from conventional techniques to biotechnology.

    Science.gov (United States)

    Rahman, Md Mahbubor; Elaissari, Abdelhamid

    2012-11-01

    Nucleic acid (DNA and RNA)-based molecular diagnosis is a promising laboratory technique because of its ability to identify disease accurately. However, one of its disadvantages is the inevitable purification and detection of nucleic acids from other contaminated entities. Different nano- and microparticles have been developed for use in an advanced, efficient high-throughput autosystem for the purification and detection of nucleic acid samples for use in molecular diagnoses. In this review, we discuss recent advances in the development of particle-based nucleic acid purification and detection. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Modeling the interplay of single-stranded binding proteins and nucleic acid secondary structure.

    Science.gov (United States)

    Forties, Robert A; Bundschuh, Ralf

    2010-01-01

    There are many important proteins which bind single-stranded nucleic acids, such as the nucleocapsid protein in HIV and the RecA DNA repair protein in bacteria. The presence of such proteins can strongly alter the secondary structure of the nucleic acid molecules. Therefore, accurate modeling of the interaction between single-stranded nucleic acids and such proteins is essential to fully understand many biological processes. We develop a model for predicting nucleic acid secondary structure in the presence of single-stranded binding proteins, and implement it as an extension of the Vienna RNA Package. All parameters needed to model nucleic acid secondary structures in the absence of proteins have been previously determined. This leaves the footprint and sequence-dependent binding affinity of the protein as adjustable parameters of our model. Using this model we are able to predict the probability of the protein binding at any position in the nucleic acid sequence, the impact of the protein on nucleic acid base pairing, the end-to-end distance distribution for the nucleic acid and FRET distributions for fluorophores attached to the nucleic acid. Source code for our modified version of the Vienna RNA package is freely available at http://bioserv.mps.ohio-state.edu/Vienna+P, implemented in C and running on Linux.

  16. BGL4 beta-glucosidase and nucleic acids encoding the same

    Science.gov (United States)

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2008-01-22

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

  17. BGL7 beta-glucosidase and nucleic acids encoding the same

    Science.gov (United States)

    Dunn-Coleman, Nigel; Ward, Michael

    2013-01-29

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

  18. BGL6 beta-glucosidase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel; Ward, Michael

    2015-08-11

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

  19. BGL6 beta-glucosidase and nucleic acids encoding the same

    Science.gov (United States)

    Dunn-Coleman, Nigel; Ward, Michael

    2014-03-04

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

  20. BGL3 beta-glucosidase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2012-10-30

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

  1. BGL7 beta-glucosidase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel; Ward, Michael

    2015-04-14

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

  2. BGL6 .beta.-glucosidase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel; Ward, Michael

    2012-10-02

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

  3. BGL7 beta-glucosidase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel; Ward, Michael

    2014-03-25

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

  4. BGL5 .beta.-glucosidase and nucleic acids encoding the same

    Science.gov (United States)

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2008-03-18

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl5, and the corresponding BGL5 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL5, recombinant BGL5 proteins and methods for producing the same.

  5. BGL6 beta-glucosidase and nucleic acids encoding the same

    Science.gov (United States)

    Dunn-Coleman, Nigel [Los Gatos, CA; Ward, Michael [San Francisco, CA

    2009-09-01

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

  6. BGL3 beta-glucosidase and nucleic acids encoding the same

    Science.gov (United States)

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2008-04-01

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

  7. BGL3 beta-glucosidase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2011-06-14

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

  8. BGL4 beta-glucosidase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2011-12-06

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

  9. Locked nucleic acid: modality, diversity, and drug discovery

    DEFF Research Database (Denmark)

    Hagedorn, Peter H.; Persson, Robert; Funder, Erik D.

    2017-01-01

    (LNA), which exhibits high binding affinity and potency, is widely used. Our understanding of RNA biology has also expanded tremendously, resulting in new approaches to engage RNA as a therapeutic target. Recent observations indicate that each oligonucleotide compound is a unique entity, and small...... structural differences between oligonucleotides can often lead to substantial differences in their pharmacological properties. Here, we outline new principles for drug discovery exploiting oligonucleotide diversity to identify rare molecules with unique pharmacological properties.......Over the past 20 years, the field of RNA-targeted therapeutics has advanced based on discoveries of modified oligonucleotide chemistries, and an ever-increasing understanding of how to apply cellular assays to identify oligonucleotides with pharmacological properties in vivo. Locked nucleic acid...

  10. Rapid genotyping using pyrene-perylene locked nucleic acid complexes

    DEFF Research Database (Denmark)

    Kumar, Santhosh T.; Myznikova, Anna; Samokhina, Evgeniya

    2013-01-01

    We have developed an assay for single strand DNA and RNA detection which is based on novel pyrene-perylene FRET pairs attached to short LNA/DNA probes. The assay is based on ratiometric emission upon binding of target DNA/RNA by three combinations of fluorescent LNA/DNA reporter strands. Specific...... is achieved with advantages of large Stokes shift (115 nm), high fluorescence quantum yields and low limit of target detection values (... geometry of the pyrene fluorophore attached to the 2'-amino group of 2'-amino-LNA in position 4 allows for the first time to efficiently utilize dipole-dipole orientation parameter for sensing of single-nucleotide polymorphisms (SNPs) in nucleic acid targets by FRET. Using novel probes, SNP detection...

  11. Multi-chamber nucleic acid amplification and detection device

    Energy Technology Data Exchange (ETDEWEB)

    Dugan, Lawrence

    2017-10-25

    A nucleic acid amplification and detection device includes an amplification cartridge with a plurality of reaction chambers for containing an amplification reagent and a visual detection reagent, and a plurality of optically transparent view ports for viewing inside the reaction chambers. The cartridge also includes a sample receiving port which is adapted to receive a fluid sample and fluidically connected to distribute the fluid sample to the reaction chamber, and in one embodiment, a plunger is carried by the cartridge for occluding fluidic communication to the reaction chambers. The device also includes a heating apparatus having a heating element which is activated by controller to generate heat when a trigger event is detected. The heating apparatus includes a cartridge-mounting section which positioned a cartridge in thermal communication with the heating element so that visual changes to the contents of the reaction chambers are viewable through the view ports.

  12. Intriguing nucleic-acid-binding features of mammalian prion protein.

    Science.gov (United States)

    Silva, Jerson L; Lima, Luís Maurício T R; Foguel, Debora; Cordeiro, Yraima

    2008-03-01

    In transmissible spongiform encephalopathies, the infectious material consists chiefly of a protein, the scrapie prion protein PrP(Sc), that carries no genetic coding material; however, prions are likely to have accomplices that chaperone their activity and promote the conversion of the cellular prion protein PrP(C) into the disease-causing isoform (PrP(Sc)). Recent studies from several laboratories indicate that PrP(C) recognizes many nucleic acids (NAs) with high affinities, and we correlate these findings with a possible pathophysiological role for this interaction. Thus, of the chaperones, NA is the most likely candidate for prions. The participation of NAs in prion propagation opens new avenues for developing new diagnostic tools and therapeutics to target prion diseases, as well as for understanding the function of PrP(C), probably as a NA chaperone.

  13. NTDB: Thermodynamic Database for Nucleic Acids, Version 2.0.

    Science.gov (United States)

    Chiu, Wing Lok Abe Kurtz; Sze, Chun Ngai; Ma, Nap Tak; Chiu, Lai Fan; Leung, Chung Wai; Au-Yeung, Steve Chik Fun

    2003-01-01

    The second release of Thermodynamic Database for Nucleic Acids, NTDB 2.0, includes more than 4600 entries (250% increase over release 1.0). It contains sequence types and details of several thermodynamic parameters (enthalpy, DeltaH; entropy, DeltaS; Gibbs free energy, DeltaG; melting temperature, T(m)), experimental models and methods for extracting thermodynamic parameters, buffer conditions as well as all relevant literature information. In addition, the database statistics and references related to NTDB are included. Information on normal and modified nucleobases and nucleosides are collected in a new section 'Nucleoside' whereby data collected thus far will be release in NTDB 2.0. The NTDB is freely available at http://ntdb.chem.cuhk.edu.hk.

  14. Nucleic acid-metal organic framework (MOF) nanoparticle conjugates.

    Science.gov (United States)

    Morris, William; Briley, William E; Auyeung, Evelyn; Cabezas, Maria D; Mirkin, Chad A

    2014-05-21

    Nanoparticles of a metal-organic framework (MOF), UiO-66-N3 (Zr6O4OH4(C8H3O4-N3)6), were synthesized. The surface of the MOF was covalently functionalized with oligonucleotides, utilizing a strain promoted click reaction between DNA appended with dibenzylcyclooctyne and azide-functionalized UiO-66-N3 to create the first MOF nanoparticle-nucleic acid conjugates. The structure of the framework was preserved throughout the chemical transformation, and the surface coverage of DNA was quantified. Due to the small pore sizes, the particles are only modified on their surfaces. When dispersed in aqueous NaCl, they exhibit increased stability and enhanced cellular uptake when compared with unfunctionalized MOF particles of comparable size.

  15. Nucleic Acid-Based Approaches for Detection of Viral Hepatitis

    Science.gov (United States)

    Behzadi, Payam; Ranjbar, Reza; Alavian, Seyed Moayed

    2014-01-01

    Context: To determining suitable nucleic acid diagnostics for individual viral hepatitis agent, an extensive search using related keywords was done in major medical library and data were collected, categorized, and summarized in different sections. Results: Various types of molecular biology tools can be used to detect and quantify viral genomic elements and analyze the sequences. These molecular assays are proper technologies for rapidly detecting viral agents with high accuracy, high sensitivity, and high specificity. Nonetheless, the application of each diagnostic method is completely dependent on viral agent. Conclusions: Despite rapidity, automation, accuracy, cost-effectiveness, high sensitivity, and high specificity of molecular techniques, each type of molecular technology has its own advantages and disadvantages. PMID:25789132

  16. Peptide nucleic acid (PNA) antisense effects in Escherichia coli

    DEFF Research Database (Denmark)

    Good, L; Nielsen, P E

    1999-01-01

    Antisense peptide nucleic acid (PNA) can be used to control cell growth, gene expression and growth phenotypes in the bacteria Escherichia coli. PNAs targeted to the RNA components of the ribosome can inhibit translation and cell growth, and PNAs targeted to mRNA can limit gene expression with gene...... and sequence specificity. In an E. coli cell extract, efficient inhibition is observed when using PNA concentrations in the nanomolar range, whereas micromolar concentrations are required for inhibition in growing cells. A mutant strain of E. coli that is more permeable to antibiotics also is more susceptible...... to antisense PNAs than the wild type. This chapter details methods for testing the antisense activities of PNA in E. coli. As an example of the specific antisense inhibition possible, we show the effects of an anti-beta-galactosidase PNA in comparison to control PNAs. With improvements in cell uptake...

  17. A novel nucleic acid analogue shows strong angiogenic activity

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Ikuko, E-mail: tukamoto@med.kagawa-u.ac.jp [Department of Pharmaco-Bio-Informatics, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki, Kita, Kagawa 761-0793 (Japan); Sakakibara, Norikazu; Maruyama, Tokumi [Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, 1314-1 Shido, Sanuki, Kagawa 769-2193 (Japan); Igarashi, Junsuke; Kosaka, Hiroaki [Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki, Kita, Kagawa 761-0793 (Japan); Kubota, Yasuo [Department of Dermatology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki, Kita, Kagawa 761-0793 (Japan); Tokuda, Masaaki [Department of Cell Physiology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki, Kita, Kagawa 761-0793 (Japan); Ashino, Hiromi [The Tokyo Metropolitan Institute of Medical Science, 1-6 Kamikitazawa2-chome, Setagaya-ku, Tokyo 156-8506 (Japan); Hattori, Kenichi; Tanaka, Shinji; Kawata, Mitsuhiro [Teikoku Seiyaku Co., Ltd., Sanbonmatsu, Higashikagawa, Kagawa 769-2695 (Japan); Konishi, Ryoji [Department of Pharmaco-Bio-Informatics, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki, Kita, Kagawa 761-0793 (Japan)

    2010-09-03

    Research highlights: {yields} A novel nucleic acid analogue (2Cl-C.OXT-A, m.w. 284) showed angiogenic potency. {yields} It stimulated the tube formation, proliferation and migration of HUVEC in vitro. {yields} 2Cl-C.OXT-A induced the activation of ERK1/2 and MEK in HUVEC. {yields} Angiogenic potency in vivo was confirmed in CAM assay and rabbit cornea assay. {yields} A synthesized small angiogenic agent would have great clinical therapeutic value. -- Abstract: A novel nucleic acid analogue (2Cl-C.OXT-A) significantly stimulated tube formation of human umbilical endothelial cells (HUVEC). Its maximum potency at 100 {mu}M was stronger than that of vascular endothelial growth factor (VEGF), a positive control. At this concentration, 2Cl-C.OXT-A moderately stimulated proliferation as well as migration of HUVEC. To gain mechanistic insights how 2Cl-C.OXT-A promotes angiogenic responses in HUVEC, we performed immunoblot analyses using phospho-specific antibodies as probes. 2Cl-C.OXT-A induced robust phosphorylation/activation of MAP kinase ERK1/2 and an upstream MAP kinase kinase MEK. Conversely, a MEK inhibitor PD98059 abolished ERK1/2 activation and tube formation both enhanced by 2Cl-C.OXT-A. In contrast, MAP kinase responses elicited by 2Cl-C.OXT-A were not inhibited by SU5416, a specific inhibitor of VEGF receptor tyrosine kinase. Collectively these results suggest that 2Cl-C.OXT-A-induces angiogenic responses in HUVEC mediated by a MAP kinase cascade comprising MEK and ERK1/2, but independently of VEGF receptor tyrosine kinase. In vivo assay using chicken chorioallantoic membrane (CAM) and rabbit cornea also suggested the angiogenic potency of 2Cl-C.OXT-A.

  18. Design of peptide-targeted liposomes containing nucleic acids.

    Science.gov (United States)

    Santos, Adriana O; da Silva, Lígia C Gomes; Bimbo, Luís M; de Lima, Maria C Pedroso; Simões, Sérgio; Moreira, João N

    2010-03-01

    Anticancer systemic gene silencing therapy has been so far limited by the inexistence of adequate carrier systems that ultimately provide an efficient intracellular delivery into target tumor cells. In this respect, one promising strategy involves the covalent attachment of internalizing-targeting ligands at the extremity of PEG chains grafted onto liposomes. Therefore, the present work aims at designing targeted liposomes containing nucleic acids, with small size, high encapsulation efficiency and able to be actively internalized by SCLC cells, using a hexapeptide (antagonist G) as a targeting ligand. For this purpose, the effect of the liposomal preparation method, loading material (ODN versus siRNA) and peptide-coupling procedure (direct coupling versus post-insertion) on each of the above-mentioned parameters was assessed. Post-insertion of DSPE-PEG-antagonist G conjugates into preformed liposomes herein named as stabilized lipid particles, resulted in targeted vesicles with a mean size of about 130 nm, encapsulation efficiency close to 100%, and a loading capacity of approximately 5 nmol siRNA/mumol of total lipid. In addition, the developed targeted vesicles showed increased internalization in SCLC cells, as well as in other tumor cells and HMEC-1 microvascular endothelial cells. The improved cellular association, however, did not correlate with enhanced downregulation of the target protein (Bcl-2) in SCLC cells. These results indicate that additional improvements need to be performed in the future, namely by ameliorating the access of the nucleic acids to the cytoplasm of the tumor cells following receptor-mediated endocytosis. Copyright 2009 Elsevier B.V. All rights reserved.

  19. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    Directory of Open Access Journals (Sweden)

    Michael G. Mauk

    2015-10-01

    Full Text Available Microfluidic components and systems for rapid (<60 min, low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs are described. A microfluidic point-of-care (POC diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1 nucleic acids (NAs are extracted from relatively large (~mL volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane” to capture sample NAs in a flow-through, filtration mode; (2 NAs captured on the membrane are isothermally (~65 °C amplified; (3 amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4 paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

  20. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Quality control material for cystic fibrosis... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a) Identification. Quality control material for cystic fibrosis nucleic acid assays. A quality control material for...

  1. Covering all the bases : Coarse-grained model design and application for nucleic acids

    NARCIS (Netherlands)

    Uusitalo, Jaakko Juhani

    2016-01-01

    Nucleic acids play a crucial role in the storage, transportation and expression of our genetic information. They have also become an interesting tool for many applications in nanotechnology. Studying biomolecular systems containing nucleic acids using experimental and imaging techniques has its

  2. Novel fluorescent nanoparticles for ultrasensitive identification of nucleic acids by optical methods

    DEFF Research Database (Denmark)

    Mulberg, Mads Westergaard; Taskova, Maria; Thomsen, Rasmus P.

    2017-01-01

    For decades, the detection of nucleic acids and their interactions at low abundances has been a challenging task. Present nucleic acid diagnostics are primarily based on enzymatic reactions including sequencing, polymerase-chain reaction and microarrays. However, the use of enzymatic amplificatio...

  3. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Science.gov (United States)

    2010-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents... panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to...: Respiratory Viral Panel Multiplex Nucleic Acid Assay;” (2) For a device that detects and identifies Human...

  4. Mesoporous Silica Nanoparticles as Carriers for Intracellular Delivery of Nucleic Acids and Subsequent Therapeutic Applications

    Directory of Open Access Journals (Sweden)

    Wenzhang Cha

    2017-05-01

    Full Text Available Nucleic acids, including DNA, microRNA (miRNA, small interfering RNA (siRNA, and antisense oligonucleotide (ASO, are powerful gene regulators, which have been demonstrated as promising drug candidates for therapeutic treatments. Nevertheless, poor cellular membrane permeability and serum stability have greatly hindered the applications of nucleic acids in biomedicine. To address these issues, associate carriers that can encapsulate and protect nucleic acids are urgently required. Mesoporous silica nanoparticles (MSNs or MSNPs, which are nanomaterials with excellent biocompatibility, large surface area for functionalization, and tunable pore size for encapsulating different cargos, are emerging as novel and ideal biomaterials for different biomedical applications. In this review paper, we focus on the applications of MSNs in nucleic acid delivery and nucleic acid-guided therapeutic treatments. General strategies for the preparation of nucleic acid-MSN complexes will be firstly introduced, followed by a summary of recent applications of MSNs in nucleic acid delivery and nucleic acid-guided therapeutics.

  5. Nucleic acids encoding mosaic clade M human immunodeficiency virus type 1 (HIV-1) envelope immunogens

    Science.gov (United States)

    Korber, Bette T; Fischer, William; Liao, Hua-Xin; Haynes, Barton F; Letvin, Norman; Hahn, Beatrice H

    2015-04-21

    The present invention relates to nucleic acids encoding mosaic clade M HIV-1 Env polypeptides and to compositions and vectors comprising same. The nucleic acids of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

  6. RIG-I detects infection with live Listeria by sensing secreted bacterial nucleic acids

    Science.gov (United States)

    Abdullah, Zeinab; Schlee, Martin; Roth, Susanne; Mraheil, Mobarak Abu; Barchet, Winfried; Böttcher, Jan; Hain, Torsten; Geiger, Sergej; Hayakawa, Yoshihiro; Fritz, Jörg H; Civril, Filiz; Hopfner, Karl-Peter; Kurts, Christian; Ruland, Jürgen; Hartmann, Gunther; Chakraborty, Trinad; Knolle, Percy A

    2012-01-01

    Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill-defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG-I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG-I-dependent IL-1β-production and inflammasome activation. The signalling molecule CARD9 contributed to IL-1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG-I provides a mechanistic explanation for efficient induction of immunity by live bacteria. PMID:23064150

  7. Nucleic Acids Research annual Database Issue and the NAR online Molecular Biology Database Collection in 2009.

    Science.gov (United States)

    Galperin, Michael Y; Cochrane, Guy R

    2009-01-01

    The current issue of Nucleic Acids Research includes descriptions of 179 databases, of which 95 are new. These databases (along with several molecular biology databases described in other journals) have been included in the Nucleic Acids Research online Molecular Biology Database Collection, bringing the total number of databases in the collection to 1170. In this introductory comment, we briefly describe some of these new databases and review the principles guiding the selection of databases for inclusion in the Nucleic Acids Research annual Database Issue and the Nucleic Acids Research online Molecular Biology Database Collection. The complete database list and summaries are available online at the Nucleic Acids Research web site (http://nar.oxfordjournals.org/).

  8. [Application status of circulating nucleic acids as biomarkers in gastric cancer].

    Science.gov (United States)

    Li, Lin; Zhang, Lianhai; Ji, Jiafu

    2014-01-01

    Considerable concentrations of circulating nucleic acids have been reported in peripheral blood from cancer patients. These circulating nucleic acids bear a variety of tumor-specific information and potentially represent a stable source of non-invasive tumor biomarkers. The assessable genetic and epigenetic changes of circulating nucleic acids include DNA mutations, copy number alterations, abnormal methylation and disruption of microRNA. Such alterations reflecting molecular characteristics of tumor tissues, provide a new clue for noninvasive, real-time and monitoring test. In the present article, the main findings of research status related to the utility of circulating nucleic acids for early diagnosis, prognosis and monitoring of gastric cancer are reviewed. In addition, the advantage, the examination technique and the application prospection of circulating nucleic acids as tumor markers are also reviewed.

  9. Nucleic Acid Extraction from Synthetic Mars Analog Soils for in situ Life Detection.

    Science.gov (United States)

    Mojarro, Angel; Ruvkun, Gary; Zuber, Maria T; Carr, Christopher E

    2017-08-01

    Biological informational polymers such as nucleic acids have the potential to provide unambiguous evidence of life beyond Earth. To this end, we are developing an automated in situ life-detection instrument that integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG) instrument. Our goal is to isolate and determine the sequence of nucleic acids from extant or preserved life on Mars, if, for example, there is common ancestry to life on Mars and Earth. As is true of metagenomic analysis of terrestrial environmental samples, the SETG instrument must isolate nucleic acids from crude samples and then determine the DNA sequence of the unknown nucleic acids. Our initial DNA extraction experiments resulted in low to undetectable amounts of DNA due to soil chemistry-dependent soil-DNA interactions, namely adsorption to mineral surfaces, binding to divalent/trivalent cations, destruction by iron redox cycling, and acidic conditions. Subsequently, we developed soil-specific extraction protocols that increase DNA yields through a combination of desalting, utilization of competitive binders, and promotion of anaerobic conditions. Our results suggest that a combination of desalting and utilizing competitive binders may establish a "universal" nucleic acid extraction protocol suitable for analyzing samples from diverse soils on Mars. Key Words: Life-detection instruments-Nucleic acids-Mars-Panspermia. Astrobiology 17, 747-760.

  10. Cell-free nucleic acids as potential markers for preeclampsia.

    Science.gov (United States)

    Hahn, S; Rusterholz, C; Hösli, I; Lapaire, O

    2011-02-01

    Preeclampsia is one of the leading causes of maternal and fetal/neonatal mortality and morbidity worldwide. Therefore, widely applicable and affordable tests are needed to make an early diagnosis before the occurrence of the clinical symptoms. Circulating cell-free nucleic acids in plasma and serum are novel biomarkers with promising clinical applications in different medical fields, including prenatal diagnosis. Quantitative changes of cell-free fetal (cff)DNA in maternal plasma as an indicator for impending preeclampsia have been reported in different studies, using real-time quantitative PCR for the male-specific SRY or DYS 14 loci. In case of early onset preeclampsia, elevated levels may be already seen in the first trimester. The increased levels of cffDNA before the onset of symptoms may be due to hypoxia/reoxygenation within the intervillous space leading to tissue oxidative stress and increased placental apoptosis and necrosis. In addition to the evidence for increased shedding of cffDNA into the maternal circulation, there is also evidence for reduced renal clearance of cffDNA in preeclampsia. As the amount of fetal DNA is currently determined by quantifying Y-chromosome specific sequences, alternative approaches such as the measurement of total cell-free DNA or the use of gender-independent fetal epigenetic markers, such as DNA methylation, offer a promising alternative. Cell-free RNA of placental origin might be another potentially useful biomarker for screening and diagnosis of preeclampsia in clinical practice. Fetal RNA is associated with subcellular placental particles that protect it from degradation. Its levels are ten-fold higher in pregnant women with preeclampsia compared to controls. In conclusion, through the use of gender-independent sequences, the universal incorporation of fetal nucleic acids into routine obstetric care and into screening or diagnostic settings using combined markers may soon become a reality. Effort has now to be put into

  11. DMPD: Plasmacytoid dendritic cells: sensing nucleic acids in viral infection andautoimmune diseases. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18641647 Plasmacytoid dendritic cells: sensing nucleic acids in viral infection andautoimmune... (.csml) Show Plasmacytoid dendritic cells: sensing nucleic acids in viral infection andautoimmune diseases.... PubmedID 18641647 Title Plasmacytoid dendritic cells: sensing nucleic acids in viral infection andautoimm...une diseases. Authors Gilliet M, Cao W, Liu YJ. Publication Nat Rev Immunol. 2008 A

  12. MINAS-a database of Metal Ions in Nucleic AcidS

    OpenAIRE

    Joachim Schnabl; Pascal Suter; Roland K. O. Sigel

    2012-01-01

    Correctly folded into the respective native 3D structure, RNA and DNA are responsible for uncountable key functions in any viable organism. In order to exert their function, metal ion cofactors are closely involved in folding, structure formation and, e.g. in ribozymes, also the catalytic mechanism. The database MINAS, Metal Ions in Nucleic AcidS (http://www.minas.uzh.ch), compiles the detailed information on innersphere, outersphere and larger coordination environment of > 70 000 metal ions ...

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

    Science.gov (United States)

    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.

  14. Nucleic Acid Extraction from Synthetic Mars Analog Soils for in situ Life Detection

    Science.gov (United States)

    Mojarro, Angel; Ruvkun, Gary; Zuber, Maria T.

    2017-01-01

    Abstract Biological informational polymers such as nucleic acids have the potential to provide unambiguous evidence of life beyond Earth. To this end, we are developing an automated in situ life-detection instrument that integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG) instrument. Our goal is to isolate and determine the sequence of nucleic acids from extant or preserved life on Mars, if, for example, there is common ancestry to life on Mars and Earth. As is true of metagenomic analysis of terrestrial environmental samples, the SETG instrument must isolate nucleic acids from crude samples and then determine the DNA sequence of the unknown nucleic acids. Our initial DNA extraction experiments resulted in low to undetectable amounts of DNA due to soil chemistry–dependent soil-DNA interactions, namely adsorption to mineral surfaces, binding to divalent/trivalent cations, destruction by iron redox cycling, and acidic conditions. Subsequently, we developed soil-specific extraction protocols that increase DNA yields through a combination of desalting, utilization of competitive binders, and promotion of anaerobic conditions. Our results suggest that a combination of desalting and utilizing competitive binders may establish a “universal” nucleic acid extraction protocol suitable for analyzing samples from diverse soils on Mars. Key Words: Life-detection instruments—Nucleic acids—Mars—Panspermia. Astrobiology 17, 747–760. PMID:28704064

  15. On-nylon membrane detection of nucleic acid molecules by rolling circle amplification.

    Science.gov (United States)

    Xu, Xinhui; Zhang, Beibei; Gan, Ping; Wu, Jian; Dai, Wei; Zhang, Ling; Wang, Jinke

    2017-09-15

    Positively-charged nylon membrane (NM) is a general solid-phase support for nucleic acid detection due to its convenient immobilization of nucleic acid materials by direct electrostatic adherence and simple UV crosslinking. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification technique for nucleic acid detection. Near-infrared fluorescence (NIRF) is a new fluorescence technique with high sensitivity due to low background. This study developed a simple method for detecting nucleic acid molecules by combining the advantages of NM, RCA and NIRF, named NIRF-based solid phase RCA on nylon membrane (NM-NIRF-sRCA). The detection system of this method only need two kinds of nucleic acid molecules: target-specific probes with a RCA primer (P) at their 3' end and a rolling circle (RC). The detection procedure consists of four steps: (1) immobilizing detected nucleic acids on NM by UV crosslinking; (2) hybridizing NM with specific probes and RC; (3) amplifying by a RCA reaction containing biotin-dUTP; (4) incubating NM with NIRF-labeled streptavidin and imaging with a NIRF imager. The method was fully testified by detecting oligonucleotides, L1 fragments of various HPV subtypes cloned in plasmid, and E.coli genomic DNA. This study thus provides a new facile method for detecting nucleic acid molecules. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Proposed Ancestors of Phage Nucleic Acid Packaging Motors (and Cells

    Directory of Open Access Journals (Sweden)

    Philip Serwer

    2011-07-01

    Full Text Available I present a hypothesis that begins with the proposal that abiotic ancestors of phage RNA and DNA packaging systems (and cells include mobile shells with an internal, molecule-transporting cavity. The foundations of this hypothesis include the conjecture that current nucleic acid packaging systems have imprints from abiotic ancestors. The abiotic shells (1 initially imbibe and later also bind and transport organic molecules, thereby providing a means for producing molecular interactions that are links in the chain of events that produces ancestors to the first molecules that are both information carrying and enzymatically active, and (2 are subsequently scaffolds on which proteins assemble to form ancestors common to both shells of viral capsids and cell membranes. Emergence of cells occurs via aggregation and merger of shells and internal contents. The hypothesis continues by using proposed imprints of abiotic and biotic ancestors to deduce an ancestral thermal ratchet-based DNA packaging motor that subsequently evolves to integrate a DNA packaging ATPase that provides a power stroke.

  17. Physical methods of nucleic acid transfer: general concepts and applications.

    Science.gov (United States)

    Villemejane, Julien; Mir, Lluis M

    2009-05-01

    Physical methods of gene (and/or drug) transfer need to combine two effects to deliver the therapeutic material into cells. The physical methods must induce reversible alterations in the plasma membrane to allow the direct passage of the molecules of interest into the cell cytosol. They must also bring the nucleic acids in contact with the permeabilized plasma membrane or facilitate access to the inside of the cell. These two effects can be achieved in one or more steps, depending upon the methods employed. In this review, we describe and compare several physical methods: biolistics, jet injection, hydrodynamic injection, ultrasound, magnetic field and electric pulse mediated gene transfer. We describe the physical mechanisms underlying these approaches and discuss the advantages and limitations of each approach as well as its potential application in research or in preclinical and clinical trials. We also provide conclusions, comparisons, and projections for future developments. While some of these methods are already in use in man, some are still under development or are used only within clinical trials for gene transfer. The possibilities offered by these methods are, however, not restricted to the transfer of genes and the complementary uses of these technologies are also discussed. As these methods of gene transfer may bypass some of the side effects linked to viral or biochemical approaches, they may find their place in specific clinical applications in the future.

  18. Nucleic acid amplification of individual molecules in a microfluidic device.

    Science.gov (United States)

    Dettloff, Roger; Yang, Esther; Rulison, Aaron; Chow, Andrea; Farinas, Javier

    2008-06-01

    A microfluidic device was developed that enabled rapid polymerase chain reaction (PCR) analysis of individual DNA molecules. The device combined a means for accessing samples serially from a microtiter plate, channels for assembling eight parallel PCR reactions, and integrated resistive heaters for rapid thermocycling (>5 degrees C/s heating, >7 degrees C/s cooling) of samples as they flowed continuously through PCR channels. Amplification was monitored by fluorescence detection of Taqman probes. The long, narrow channels (10 microm x 180 microm x 40 mm) allowed sufficient separation between neighboring DNA templates to enable amplification of discreet DNA molecules. The functionality of the device was demonstrated by reproducibly amplifying a 2D6.6 CYP450 template and distinguishing between wild-type and mutant sequences using Taqman probes. A comparison of the rate of individual amplification events to the expected Poisson distribution confirmed that the device could reliably analyze individual DNA molecules. This work establishes the feasibility of rapid, single-molecule interrogation of nucleic acids.

  19. Monitoring Gene Expression In Vivo with Nucleic Acid Molecular Switches

    Energy Technology Data Exchange (ETDEWEB)

    David C. Ward; Patricia Bray-Ward

    2005-01-26

    The overall objectives of this project were (1) to develop allosteric ribozymes capable of acting as molecular switches for monitoring the levels of both wild-type and mutant mRNA species in living cells and whole animals and (2) to develop highly efficient reagents to deliver nucleic acid molecular switches into living cells, tissues and animals with the ultimate goal of expression profiling specific mRNAs of diagnostic or prognostic value within tumors in animals. During the past year, we have moved our laboratory to Nevada and in the moving process we have lost electronic and paper copies of prior progress reports concerning the construction and biological properties of the molecular switches. Since there was minimal progress during the last year on molecular switches, we are relying on past project reports to provide a summary of our data on this facet of the grant. Here we are summarizing the work done on the delivery reagents and their application to inducing mutations in living cells, which will include work done during the no cost extension.

  20. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    Science.gov (United States)

    Mauk, Michael G.; Liu, Changchun; Song, Jinzhao; Bau, Haim H.

    2015-01-01

    Microfluidic components and systems for rapid (nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane”) to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed. PMID:27600235

  1. Development of a Capillary-driven, Microfluidic, Nucleic Acid Biosensor

    Directory of Open Access Journals (Sweden)

    Fei HE

    2011-12-01

    Full Text Available An ideal point-of-care device would incorporate the simplicity and reliability of a lateral flow assay with a microfluidic device. Our system consists of self-priming microfluidics with sealed conjugate pads of reagent delivery and an absorbent pad for additional fluid draw. Using poly (methyl methacrylate (PMMA as a substrate, we have developed a single-step surface modification method which allows strong capillary flow within a sealed microchannel. Conjugate pads within the device held trapped complex consisting of the magnetic beads and nucleic-acid-probe-conjugated horseradish peroxidase (HRP. Magnetic beads were released when sample entered the chamber and hybridized with the complex. The complex was immobilized over a magnet while a luminol co-reactant stream containing H2O2 was merged with the channel. A plate reader was able to quantify the chemiluminescence signal. This new format of biosensor will allow for a smaller and more sensitive biosensor, as well as commercial-scale manufacturing and low materials cost.

  2. Application of locked nucleic acid-based probes in fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Fontenete, Sílvia; Carvalho, Daniel R; Guimarães, Nuno

    2016-01-01

    of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2′-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall......Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2′-O-methyl (2′-OMe) RNA modifications have...

  3. Double-helical nucleic acids with cross-linked strands: synthesis and applications in molecular biology

    Energy Technology Data Exchange (ETDEWEB)

    Antsypovitch, Sergei I; Oretskaya, Tat' yana S [Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow (Russian Federation)

    1998-03-31

    Data on the methods employed for cross-linking of DNA strands and for the synthesis of oligonucleotide duplexes with cross-links between strands are summarised. Existing methods are systematised; their advantages and drawbacks are discussed. The examples of applications of DNA duplexes with covalently cross-linked chains for the study of protein-nucleic acid recognition and mechanisms of action of nucleic acid-binding proteins for gaining information about the spatial structure of nucleic acids, and for the solution of other problems of molecular biology are given. The bibliography includes 131 references.

  4. Theranostic Nucleic Acid Binding Nanoprobe Exerts Anti-inflammatory and Cytoprotective Effects in Ischemic Injury.

    Science.gov (United States)

    Chen, Howard H; Yuan, Hushan; Cho, Hoonsung; Feng, Yan; Ngoy, Soeun; Kumar, Anand T N; Liao, Ronglih; Chao, Wei; Josephson, Lee; Sosnovik, David E

    2017-01-01

    Extracellular nucleic acids are proinflammatory molecules that have been implicated in a diverse range of diseases. We report here the development of a multivalent nucleic acid scavenging nanoprobe, where the fluorochrome thiazole orange (TO) is conjugated to a polymeric 40 kDa dextran carrier. Dextran-TO (Dex-TO) has nanomolar affinity for mammalian and bacterial nucleic acids and attenuates the production of inflammatory cytokines from activated macrophages exposed to DNA and RNA. Mice with myocardial ischemia reperfusion that were treated with Dex-TO showed a decrease in myocardial macrophage infiltration at 24 hours (ptheranostic) utility in a broad range of conditions including ischemia, trauma, burns, sepsis and autoimmune disease.

  5. The third annual BRDS on research and development of nucleic acid-based nanomedicines.

    Science.gov (United States)

    Chaudhary, Amit Kumar; Mahato, Ram I

    2017-02-01

    The completion of human genome project, decrease in the sequencing cost, and correlation of genome sequencing data with specific diseases led to the exponential rise in the nucleic acid-based therapeutic approaches. In the third annual Biopharmaceutical Research and Development Symposium (BRDS) held at the Center for Drug Discovery and Lozier Center for Pharmacy Sciences and Education at the University of Nebraska Medical Center (UNMC), we highlighted the remarkable features of the nucleic acid-based nanomedicines, their significance, NIH funding opportunities on nanomedicines and gene therapy research, challenges and opportunities in the clinical translation of nucleic acids into therapeutics, and the role of intellectual property (IP) in drug discovery and development.

  6. MINAS--a database of Metal Ions in Nucleic AcidS.

    Science.gov (United States)

    Schnabl, Joachim; Suter, Pascal; Sigel, Roland K O

    2012-01-01

    Correctly folded into the respective native 3D structure, RNA and DNA are responsible for uncountable key functions in any viable organism. In order to exert their function, metal ion cofactors are closely involved in folding, structure formation and, e.g. in ribozymes, also the catalytic mechanism. The database MINAS, Metal Ions in Nucleic AcidS (http://www.minas.uzh.ch), compiles the detailed information on innersphere, outersphere and larger coordination environment of >70,000 metal ions of 36 elements found in >2000 structures of nucleic acids contained today in the PDB and NDB. MINAS is updated monthly with new structures and offers a multitude of search functions, e.g. the kind of metal ion, metal-ligand distance, innersphere and outersphere ligands defined by element or functional group, residue, experimental method, as well as PDB entry-related information. The results of each search can be saved individually for later use with so-called miniPDB files containing the respective metal ion together with the coordination environment within a 15 Å radius. MINAS thus offers a unique way to explore the coordination geometries and ligands of metal ions together with the respective binding pockets in nucleic acids.

  7. Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

    Science.gov (United States)

    Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

    2015-02-01

    We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. The effects of borate minerals on the synthesis of nucleic acid bases, amino acids and biogenic carboxylic acids from formamide.

    Science.gov (United States)

    Saladino, Raffaele; Barontini, Maurizio; Cossetti, Cristina; Di Mauro, Ernesto; Crestini, Claudia

    2011-08-01

    The thermal condensation of formamide in the presence of mineral borates is reported. The products afforded are precursors of nucleic acids, amino acids derivatives and carboxylic acids. The efficiency and the selectivity of the reaction was studied in relation to the elemental composition of the 18 minerals analyzed. The possibility of synthesizing at the same time building blocks of both genetic and metabolic apparatuses, along with the production of amino acids, highlights the interest of the formamide/borate system in prebiotic chemistry.

  9. Gene Therapy for Advanced Melanoma: Selective Targeting and Therapeutic Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Joana R. Viola

    2013-01-01

    Full Text Available Despite recent advances, the treatment of malignant melanoma still results in the relapse of the disease, and second line treatment mostly fails due to the occurrence of resistance. A wide range of mutations are known to prevent effective treatment with chemotherapeutic drugs. Hence, approaches with biopharmaceuticals including proteins, like antibodies or cytokines, are applied. As an alternative, regimens with therapeutically active nucleic acids offer the possibility for highly selective cancer treatment whilst avoiding unwanted and toxic side effects. This paper gives a brief introduction into the mechanism of this devastating disease, discusses the shortcoming of current therapy approaches, and pinpoints anchor points which could be harnessed for therapeutic intervention with nucleic acids. We bring the delivery of nucleic acid nanopharmaceutics into perspective as a novel antimelanoma therapeutic approach and discuss the possibilities for melanoma specific targeting. The latest reports on preclinical and already clinical application of nucleic acids in melanoma are discussed.

  10. Polycation cytotoxicity: a delicate matter for nucleic acid therapy-focus on polyethylenimine

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Larsen, Anna K.; Hunter, A. Christy

    2010-01-01

    This article provides a critical assessment of the major challenges facing nucleic acid therapeutics, focusing on the safety and efficacy of delivery strategies using synthetic polycations and particularly polyethylenimines. Deficiencies in the field and avenues for further research are identified...

  11. Antibacterial Peptide Nucleic Acid-Antimicrobial Peptide (PNA-AMP) Conjugates

    DEFF Research Database (Denmark)

    Hansen, Anna Mette; Bonke, Gitte; Larsen, Camilla Josephine

    2016-01-01

    Antisense peptide nucleic acid (PNA) oligomers constitute a novel class of potential antibiotics that inhibit bacterial growth via specific knockdown of essential gene expression. However, discovery of efficient, nontoxic delivery vehicles for such PNA oligomers has remained a challenge...

  12. Nucleic acid polymeric properties and electrostatics: Directly comparing theory and simulation with experiment.

    Science.gov (United States)

    Sim, Adelene Y L

    2016-06-01

    Nucleic acids are biopolymers that carry genetic information and are also involved in various gene regulation functions such as gene silencing and protein translation. Because of their negatively charged backbones, nucleic acids are polyelectrolytes. To adequately understand nucleic acid folding and function, we need to properly describe its i) polymer/polyelectrolyte properties and ii) associating ion atmosphere. While various theories and simulation models have been developed to describe nucleic acids and the ions around them, many of these theories/simulations have not been well evaluated due to complexities in comparison with experiment. In this review, I discuss some recent experiments that have been strategically designed for straightforward comparison with theories and simulation models. Such data serve as excellent benchmarks to identify limitations in prevailing theories and simulation parameters. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. The 2015 Nucleic Acids Research Database Issue and molecular biology database collection

    National Research Council Canada - National Science Library

    Galperin, Michael Y; Rigden, Daniel J; Fernández-Suárez, Xosé M

    2015-01-01

    The 2015 Nucleic Acids Research Database Issue contains 172 papers that include descriptions of 56 new molecular biology databases, and updates on 115 databases whose descriptions have been previously...

  14. Nucleic acid sensing and innate immunity: signaling pathways controlling viral pathogenesis and autoimmunity

    Science.gov (United States)

    Ahlers, Laura R. H.; Goodman, Alan G.

    2016-01-01

    Innate immunity refers to the body’s initial response to curb infection upon exposure to invading organisms. While the detection of pathogen-associated molecules is an ancient form of host defense, if dysfunctional, autoimmune disease may result. The innate immune response during pathogenic infection is initiated through the activation of receptors recognizing conserved molecular patterns, such as nucleic acids from a virus’ genome or replicative cycle. Additionally, the host’s own nucleic acids are capable of activating an immune response. Therefore, it follows that the nucleic acid-sensing pathways must be tightly controlled to avoid an autoimmune response from recognition of self, yet still be unimpeded to respond to viral infections. In this review, we will describe the nucleic acid sensing pathways and how they respond to virus infection. Moreover, we will discuss autoimmune diseases that develop when these pathways fail to signal properly and identify knowledge gaps that are prime for interrogation. PMID:27857881

  15. Beyond H&E: integration of nucleic acid-based analyses into diagnostic pathology.

    Science.gov (United States)

    Maes, R K; Langohr, I M; Wise, A G; Smedley, R C; Thaiwong, T; Kiupel, M

    2014-01-01

    Veterinary pathology of infectious, particularly viral, and neoplastic diseases has advanced significantly with the advent of newer molecular methodologies that can detect nucleic acid of infectious agents within microscopic lesions, differentiate neoplastic from nonneoplastic cells, or determine the suitability of a targeted therapy by detecting specific mutations in certain cancers. Polymerase chain reaction-based amplification of DNA or RNA and in situ hybridization are currently the most commonly used methods for nucleic acid detection. In contrast, the main methodology used for protein detection within microscopic lesions is immunohistochemistry. Other methods that allow for analysis of nucleic acids within a particular cell type or individual cells, such as laser capture microdissection, are also available in some laboratories. This review gives an overview of the factors that influence the accurate analysis of nucleic acids in formalin-fixed tissues, as well as of different approaches to detect such targets.

  16. THE VARIATION OF NUCLEIC ACIDS CONTENT AFTER SIMAZIN TREATMENT ON VICIA SATIVA L.

    Directory of Open Access Journals (Sweden)

    Odeta Grama-Tiganasu

    2005-08-01

    Full Text Available Simazin has in certain conditions stimulatory effects on nucleic acids biosynthese. The biosyntese and mitotic division stimulation sugest the possibility to use simazin like growing and germination stimulator.

  17. Nucleic Acid Extraction from Synthetic Mars Analog Soils for in situ Life Detection

    Science.gov (United States)

    Mojarro, Angel; Ruvkun, Gary; Zuber, Maria T.; Carr, Christopher E.

    2017-08-01

    Biological informational polymers such as nucleic acids have the potential to provide unambiguous evidence of life beyond Earth. To this end, we are developing an automated in situ life-detection instrument that integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG) instrument. Our goal is to isolate and determine the sequence of nucleic acids from extant or preserved life on Mars, if, for example, there is common ancestry to life on Mars and Earth. As is true of metagenomic analysis of terrestrial environmental samples, the SETG instrument must isolate nucleic acids from crude samples and then determine the DNA sequence of the unknown nucleic acids. Our initial DNA extraction experiments resulted in low to undetectable amounts of DNA due to soil chemistry-dependent soil-DNA interactions, namely adsorption to mineral surfaces, binding to divalent/trivalent cations, destruction by iron redox cycling, and acidic conditions. Subsequently, we developed soil-specific extraction protocols that increase DNA yields through a combination of desalting, utilization of competitive binders, and promotion of anaerobic conditions. Our results suggest that a combination of desalting and utilizing competitive binders may establish a "universal" nucleic acid extraction protocol suitable for analyzing samples from diverse soils on Mars.

  18. DSSR-enhanced visualization of nucleic acid structures in Jmol.

    Science.gov (United States)

    Hanson, Robert M; Lu, Xiang-Jun

    2017-05-03

    Sophisticated and interactive visualizations are essential for making sense of the intricate 3D structures of macromolecules. For proteins, secondary structural components are routinely featured in molecular graphics visualizations. However, the field of RNA structural bioinformatics is still lagging behind; for example, current molecular graphics tools lack built-in support even for base pairs, double helices, or hairpin loops. DSSR (Dissecting the Spatial Structure of RNA) is an integrated and automated command-line tool for the analysis and annotation of RNA tertiary structures. It calculates a comprehensive and unique set of features for characterizing RNA, as well as DNA structures. Jmol is a widely used, open-source Java viewer for 3D structures, with a powerful scripting language. JSmol, its reincarnation based on native JavaScript, has a predominant position in the post Java-applet era for web-based visualization of molecular structures. The DSSR-Jmol integration presented here makes salient features of DSSR readily accessible, either via the Java-based Jmol application itself, or its HTML5-based equivalent, JSmol. The DSSR web service accepts 3D coordinate files (in mmCIF or PDB format) initiated from a Jmol or JSmol session and returns DSSR-derived structural features in JSON format. This seamless combination of DSSR and Jmol/JSmol brings the molecular graphics of 3D RNA structures to a similar level as that for proteins, and enables a much deeper analysis of structural characteristics. It fills a gap in RNA structural bioinformatics, and is freely accessible (via the Jmol application or the JSmol-based website http://jmol.x3dna.org). © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. A modern mode of activation for nucleic acid enzymes.

    Directory of Open Access Journals (Sweden)

    Dominique Lévesque

    2007-07-01

    Full Text Available Through evolution, enzymes have developed subtle modes of activation in order to ensure the sufficiently high substrate specificity required by modern cellular metabolism. One of these modes is the use of a target-dependent module (i.e. a docking domain such as those found in signalling kinases. Upon the binding of the target to a docking domain, the substrate is positioned within the catalytic site. The prodomain acts as a target-dependent module switching the kinase from an off state to an on state. As compared to the allosteric mode of activation, there is no need for the presence of a third partner. None of the ribozymes discovered to date have such a mode of activation, nor does any other known RNA. Starting from a specific on/off adaptor for the hepatitis delta virus ribozyme, that differs but has a mechanism reminiscent of this signalling kinase, we have adapted this mode of activation, using the techniques of molecular engineering, to both catalytic RNAs and DNAs exhibiting various activities. Specifically, we adapted three cleaving ribozymes (hepatitis delta virus, hammerhead and hairpin ribozymes, a cleaving 10-23 deoxyribozyme, a ligating hairpin ribozyme and an artificially selected capping ribozyme. In each case, there was a significant gain in terms of substrate specificity. Even if this mode of control is unreported for natural catalytic nucleic acids, its use needs not be limited to proteinous enzymes. We suggest that the complexity of the modern cellular metabolism might have been an important selective pressure in this evolutionary process.

  20. Toward Complete Sequence Flexibility of Nucleic Acid Base Analogue FRET.

    Science.gov (United States)

    Wranne, Moa S; Füchtbauer, Anders Foller; Dumat, Blaise; Bood, Mattias; El-Sagheer, Afaf H; Brown, Tom; Gradén, Henrik; Grøtli, Morten; Wilhelmsson, L Marcus

    2017-07-12

    Förster resonance energy transfer (FRET) using fluorescent base analogues is a powerful means of obtaining high-resolution nucleic acid structure and dynamics information that favorably complements techniques such as NMR and X-ray crystallography. Here, we expand the base-base FRET repertoire with an adenine analogue FRET-pair. Phosphoramidite-protected quadracyclic 2'-deoxyadenosine analogues qAN1 (donor) and qAnitro (acceptor) were synthesized and incorporated into DNA by a generic, reliable, and high-yielding route, and both constitute excellent adenine analogues. The donor, qAN1, has quantum yields reaching 21% and 11% in single- and double-strands, respectively. To the best of our knowledge, this results in the highest average brightness of an adenine analogue inside DNA. Its potent emissive features overlap well with the absorption of qAnitro and thus enable accurate FRET-measurements over more than one turn of B-DNA. As we have shown previously for our cytosine analogue FRET-pair, FRET between qAN1 and qAnitro positioned at different base separations inside DNA results in efficiencies that are highly dependent on both distance and orientation. This facilitates significantly enhanced resolution in FRET structure determinations, demonstrated here in a study of conformational changes of DNA upon binding of the minor groove binder netropsin. Finally, we note that the donor and acceptor of our cytosine FRET-pair, tC(O) and tCnitro, can be conveniently combined with the acceptor and donor of our current adenine pair, respectively. Consequently, our base analogues can now measure base-base FRET between 3 of the 10 possible base combinations and, through base-complementarity, between all sequence positions in a duplex.

  1. Polarizable model potential function for nucleic acid bases.

    Science.gov (United States)

    Nakagawa, Setsuko

    2007-07-15

    A polarizable model potential (PMP) function for adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) is developed on the basis of ab initio molecular orbital calculations at the MP2/6-31+G* level. The PMP function consists of Coulomb, van der Waals, and polarization terms. The permanent atomic charges of the Coulomb term are determined by using electrostatic potential (ESP) optimization. The multicenter polarizabilities of the polarization term are determined by using polarized one-electron potential (POP) optimization in which the electron density changes induced by a test charge are target. Isotropic and anisotropic polarizabilities are adopted as the multicenter polarizabilities. In the PMP calculations using the optimized parameters, the interaction energies of Watson-Crick type A-T and C-G base pairs were -15.6 and -29.4 kcal/mol, respectively. The interaction energy of Hoogsteen type A-T base pair was -17.8 kcal/mol. These results reproduce well the quantum chemistry calculations at the MP2/6-311++G(3df,2pd) level within the differences of 0.6 kcal/mol. The stacking energies of A-T and C-G were -9.7 and -10.9 kcal/mol. These reproduce well the calculation results at the MP2/6-311++G (2d,2p) level within the differences of 1.3 kcal/mol. The potential energy surfaces of the system in which a sodium ion or a chloride ion is adjacent to the nucleic acid base are calculated. The interaction energies of the PMP function reproduced well the calculation results at the MP2/6-31+G* or MP2/6-311++G(2d,2p) level. The reason why the PMP function reproduces well the high-level quantum mechanical interaction energies is addressed from the viewpoint of each energy terms. Copyright (c) 2007 Wiley Periodicals, Inc.

  2. Analysis of protein-nucleic acid interactions by photochemical cross-linking and mass spectrometry

    DEFF Research Database (Denmark)

    Steen, Hanno; Jensen, Ole Nørregaard

    2002-01-01

    and for sequencing of peptide-nucleic acid heteroconjugates. The combination of photochemical cross-linking and MS provides a fast screening method to gain insights into the overall structure and formation of protein-oligonucleotide complexes. Because the analytical methods are continuously refined and protein...... structural data are rapidly accumulating in databases, we envision that many protein-nucleic acid assemblies will be initially characterized by combinations of cross-linking methods, MS, and computational molecular modeling....

  3. Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

    DEFF Research Database (Denmark)

    Crouzier, Lucile; Dubois, Camille; Wengel, Jesper

    2012-01-01

    Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving...... LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI....

  4. Microfluidic devices for nucleic acid (NA) isolation, isothermal NA amplification, and real-time detection.

    Science.gov (United States)

    Mauk, Michael G; Liu, Changchun; Sadik, Mohamed; Bau, Haim H

    2015-01-01

    Molecular (nucleic acid)-based diagnostics tests have many advantages over immunoassays, particularly with regard to sensitivity and specificity. Most on-site diagnostic tests, however, are immunoassay-based because conventional nucleic acid-based tests (NATs) require extensive sample processing, trained operators, and specialized equipment. To make NATs more convenient, especially for point-of-care diagnostics and on-site testing, a simple plastic microfluidic cassette ("chip") has been developed for nucleic acid-based testing of blood, other clinical specimens, food, water, and environmental samples. The chip combines nucleic acid isolation by solid-phase extraction; isothermal enzymatic amplification such as LAMP (Loop-mediated AMPlification), NASBA (Nucleic Acid Sequence Based Amplification), and RPA (Recombinase Polymerase Amplification); and real-time optical detection of DNA or RNA analytes. The microfluidic cassette incorporates an embedded nucleic acid binding membrane in the amplification reaction chamber. Target nucleic acids extracted from a lysate are captured on the membrane and amplified at a constant incubation temperature. The amplification product, labeled with a fluorophore reporter, is excited with a LED light source and monitored in situ in real time with a photodiode or a CCD detector (such as available in a smartphone). For blood analysis, a companion filtration device that separates plasma from whole blood to provide cell-free samples for virus and bacterial lysis and nucleic acid testing in the microfluidic chip has also been developed. For HIV virus detection in blood, the microfluidic NAT chip achieves a sensitivity and specificity that are nearly comparable to conventional benchtop protocols using spin columns and thermal cyclers.

  5. Nucleic acid sensing pattern recognition receptors in the development of colorectal cancer and colitis.

    Science.gov (United States)

    He, Liangmei; Chen, Yayun; Wu, Yuanbing; Xu, Ying; Zhang, Zixiang; Liu, Zhiping

    2017-07-01

    Colorectal cancer (CRC) is a leading cause of cancer-related deaths that is often associated with inflammation initiated by activation of pattern recognition receptors (PRRs). Nucleic acid sensing PRRs are one of the major subsets of PRRs that sense nucleic acid (DNA and RNA), mainly including some members of Toll-like receptors (TLR3, 7, 8, 9), AIM2-like receptors (AIM2, IFI16), STING, cGAS, RNA polymerase III, and DExD/H box nucleic acid helicases (such as RIG-I like receptors (RIG-I, MDA5, LPG2), DDX1, 3, 5, 7, 17, 21, 41, 60, and DHX9, 36). Activation of these receptors eventually leads to the release of cytokines and activation of immune cells, which are well known to play crucial roles in host defense against intracellular bacterial and virus infection. However, the functions of these nucleic acid sensing PRRs in the other diseases such as CRC and colitis remain largely unknown. Recent studies indicated that nucleic acid sensing PRRs contribute to CRC and/or colitis development, and therapeutic modulation of nucleic acid sensing PRRs may reduce the risk of CRC development. However, until now, a comprehensive review on the role of nucleic acid sensing PRRs in CRC and colitis is still lacking. This review provided an overview of the roles as well as the mechanisms of these nucleic acid sensing PRRs (AIM2, STING, cGAS, RIG-I and its downstream molecules, DDX3, 5, 6,17, and DHX9, 36) in CRC and colitis, which may aid the diagnosis, therapy, and prognostic prediction of CRC and colitis.

  6. Development of Chemiluminescent Lateral Flow Assay for the Detection of Nucleic Acids

    OpenAIRE

    Sam R. Nugen; Catherine Fill; Yuhong Wang

    2012-01-01

    Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety. Herein we report the development of a nucleic acid sequence-based lateral flow assay which achieves a low limit of detection using chemiluminescence. On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level. To demonstrate this assay, we detected synthetic nucleic acid sequences representative of Trypanosoma mRNA, th...

  7. Peptide Nucleic Acids Having Enhanced Binding Affinity, Sequence Specificity and Solubility

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from a group consisting of naturally......-occurring nucleobases and non-naturally-occurring nucleobases attached to a polyamide backbone, and contain C1-C8 alkylamine side chains. Methods of enhancing the solubility, binding affinity and sequence specificity of PNAs are provided....

  8. Features of "All LNA" Duplexes Showing a New Type of Nucleic Acid Geometry.

    Science.gov (United States)

    Förster, Charlotte; Eichert, André; Oberthür, Dominik; Betzel, Christian; Geßner, Reinhard; Nitsche, Andreas; Fürste, Jens P

    2012-01-01

    "Locked nucleic acids" (LNAs) belong to the backbone-modified nucleic acid family. The 2'-O,4'-C-methylene-β-D-ribofuranose nucleotides are used for single or multiple substitutions in RNA molecules and thereby introduce enhanced bio- and thermostability. This renders LNAs powerful tools for diagnostic and therapeutic applications. RNA molecules maintain the overall canonical A-type conformation upon substitution of single or multiple residues/nucleotides by LNA monomers. The structures of "all" LNA homoduplexes, however, exhibit significant differences in their overall geometry, in particular a decreased twist, roll and propeller twist. This results in a widening of the major groove, a decrease in helical winding, and an enlarged helical pitch. Therefore, the LNA duplex structure can no longer be described as a canonical A-type RNA geometry but can rather be brought into proximity to other backbone-modified nucleic acids, like glycol nucleic acids or peptide nucleic acids. LNA-modified nucleic acids provide thus structural and functional features that may be successfully exploited for future application in biotechnology and drug discovery.

  9. Nucleic acid purification from plants, animals and microbes in under 30 seconds.

    Directory of Open Access Journals (Sweden)

    Yiping Zou

    2017-11-01

    Full Text Available Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling, means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries.

  10. Logic gates and antisense DNA devices operating on a translator nucleic Acid scaffold.

    Science.gov (United States)

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

    2009-07-28

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

  11. Features of “All LNA” Duplexes Showing a New Type of Nucleic Acid Geometry

    Directory of Open Access Journals (Sweden)

    Charlotte Förster

    2012-01-01

    Full Text Available “Locked nucleic acids” (LNAs belong to the backbone-modified nucleic acid family. The 2′-O,4′-C-methylene-β-D-ribofuranose nucleotides are used for single or multiple substitutions in RNA molecules and thereby introduce enhanced bio- and thermostability. This renders LNAs powerful tools for diagnostic and therapeutic applications. RNA molecules maintain the overall canonical A-type conformation upon substitution of single or multiple residues/nucleotides by LNA monomers. The structures of “all” LNA homoduplexes, however, exhibit significant differences in their overall geometry, in particular a decreased twist, roll and propeller twist. This results in a widening of the major groove, a decrease in helical winding, and an enlarged helical pitch. Therefore, the LNA duplex structure can no longer be described as a canonical A-type RNA geometry but can rather be brought into proximity to other backbone-modified nucleic acids, like glycol nucleic acids or peptide nucleic acids. LNA-modified nucleic acids provide thus structural and functional features that may be successfully exploited for future application in biotechnology and drug discovery.

  12. Highly Efficient Synthesis of Allopurinol Locked Nucleic Acid Monomer by C6 Deamination of 8-Aza-7-bromo-7-deazaadenine Locked Nucleic Acid Monomer

    DEFF Research Database (Denmark)

    Kosbar, Tamer Reda El-Saeed; Sofan, M.; Abou-Zeid, L.

    2013-01-01

    An allopurinol locked nucleic acid (LNA) monomer was prepared by a novel strategy through C6 deamination of the corresponding 8-aza-7-bromo-7-deazaadenine LNA monomer with aqueous sodium hydroxide. An 8-aza-7-deazaadenine LNA monomer was also synthesized by a modification of the new synthetic...... the required LNA monomers....

  13. DNA-like double helix formed by peptide nucleic acid

    DEFF Research Database (Denmark)

    Wittung, P; Nielsen, Peter E.; Buchardt, O

    1994-01-01

    Although the importance of the nucleobases in the DNA double helix is well understood, the evolutionary significance of the deoxyribose phosphate backbone and the contribution of this chemical entity to the overall helical structure and stability of the double helix is not so clear. Peptide nucleic...

  14. NAIL: Nucleic Acid detection using Isotachophoresis and Loop-mediated isothermal amplification.

    Science.gov (United States)

    Borysiak, Mark D; Kimura, Kevin W; Posner, Jonathan D

    2015-04-07

    Nucleic acid amplification tests are the gold standard for many infectious disease diagnoses due to high sensitivity and specificity, rapid operation, and low limits of detection. Despite the advantages of nucleic acid amplification tests, they currently offer limited point-of-care (POC) utility due to the need for complex instruments and laborious sample preparation. We report the development of the Nucleic Acid Isotachophoresis LAMP (NAIL) diagnostic device. NAIL uses isotachophoresis (ITP) and loop-mediated isothermal amplification (LAMP) to extract and amplify nucleic acids from complex matrices in less than one hour inside of an integrated chip. ITP is an electrokinetic separation technique that uses an electric field and two buffers to extract and purify nucleic acids in a single step. LAMP amplifies nucleic acids at constant temperature and produces large amounts of DNA that can be easily detected. A mobile phone images the amplification results to eliminate the need for laser fluorescent detection. The device requires minimal user intervention because capillary valves and heated air chambers act as passive valves and pumps for automated fluid actuation. In this paper, we describe NAIL device design and operation, and demonstrate the extraction and detection of pathogenic E. coli O157:H7 cells from whole milk samples. We use the Clinical and Laboratory Standards Institute (CLSI) limit of detection (LoD) definitions that take into account the variance from both positive and negative samples to determine the diagnostic LoD. According to the CLSI definition, the NAIL device has a limit of detection (LoD) of 1000 CFU mL(-1) for E. coli cells artificially inoculated into whole milk, which is two orders of magnitude improvement to standard tube-LAMP reactions with diluted milk samples and comparable to lab-based methods. The NAIL device potentially offers significant reductions in the complexity and cost of traditional nucleic acid diagnostics for POC applications.

  15. Optimizing scoring function of protein-nucleic acid interactions with both affinity and specificity.

    Directory of Open Access Journals (Sweden)

    Zhiqiang Yan

    Full Text Available Protein-nucleic acid (protein-DNA and protein-RNA recognition is fundamental to the regulation of gene expression. Determination of the structures of the protein-nucleic acid recognition and insight into their interactions at molecular level are vital to understanding the regulation function. Recently, quantitative computational approach has been becoming an alternative of experimental technique for predicting the structures and interactions of biomolecular recognition. However, the progress of protein-nucleic acid structure prediction, especially protein-RNA, is far behind that of the protein-ligand and protein-protein structure predictions due to the lack of reliable and accurate scoring function for quantifying the protein-nucleic acid interactions. In this work, we developed an accurate scoring function (named as SPA-PN, SPecificity and Affinity of the Protein-Nucleic acid interactions for protein-nucleic acid interactions by incorporating both the specificity and affinity into the optimization strategy. Specificity and affinity are two requirements of highly efficient and specific biomolecular recognition. Previous quantitative descriptions of the biomolecular interactions considered the affinity, but often ignored the specificity owing to the challenge of specificity quantification. We applied our concept of intrinsic specificity to connect the conventional specificity, which circumvents the challenge of specificity quantification. In addition to the affinity optimization, we incorporated the quantified intrinsic specificity into the optimization strategy of SPA-PN. The testing results and comparisons with other scoring functions validated that SPA-PN performs well on both the prediction of binding affinity and identification of native conformation. In terms of its performance, SPA-PN can be widely used to predict the protein-nucleic acid structures and quantify their interactions.

  16. Features of “All LNA” Duplexes Showing a New Type of Nucleic Acid Geometry

    OpenAIRE

    Charlotte Förster; André Eichert; Dominik Oberthür; Christian Betzel; Reinhard Geßner; Andreas Nitsche; Fürste, Jens P.

    2012-01-01

    “Locked nucleic acids” (LNAs) belong to the backbone-modified nucleic acid family. The 2′-O,4′-C-methylene- β -D-ribofuranose nucleotides are used for single or multiple substitutions in RNA molecules and thereby introduce enhanced bio- and thermostability. This renders LNAs powerful tools for diagnostic and therapeutic applications. RNA molecules maintain the overall canonical A-type conformation upon substitution of single or multiple residues/nucleotides by LNA monomers. The structures of ...

  17. Nucleic acid-induced antiviral immunity in invertebrates: an evolutionary perspective.

    Science.gov (United States)

    Wang, Pei-Hui; Weng, Shao-Ping; He, Jian-Guo

    2015-02-01

    Nucleic acids derived from viral pathogens are typical pathogen associated molecular patterns (PAMPs). In mammals, the recognition of viral nucleic acids by pattern recognition receptors (PRRs), which include Toll-like receptors (TLRs) and retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), induces the release of inflammatory cytokines and type I interferons (IFNs) through the activation of nuclear factor κB (NF-κB) and interferon regulatory factor (IRF) 3/7 pathways, triggering the host antiviral state. However, whether nucleic acids can induce similar antiviral immunity in invertebrates remains ambiguous. Several studies have reported that nucleic acid mimics, especially dsRNA mimic poly(I:C), can strongly induce non-specific antiviral immune responses in insects, shrimp, and oyster. This behavior shows multiple similarities to the hallmarks of mammalian IFN responses. In this review, we highlight the current understanding of nucleic acid-induced antiviral immunity in invertebrates. We also discuss the potential recognition and regulatory mechanisms that confer non-specific antiviral immunity on invertebrate hosts. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Efficacy Assessment of Nucleic Acid Decontamination Reagents Used in Molecular Diagnostic Laboratories.

    Science.gov (United States)

    Fischer, Melina; Renevey, Nathalie; Thür, Barbara; Hoffmann, Donata; Beer, Martin; Hoffmann, Bernd

    2016-01-01

    The occurrence of nucleic acid cross contamination in the laboratory resulting in false positive results of diagnostic samples is seriously problematic. Despite precautions to minimize or even avoid nucleic acid cross contaminations, it may appear anyway. Until now, no standardized strategy is available to evaluate the efficacy of commercially offered decontamination reagents. Therefore, a protocol for the reliable determination of nucleic acid decontamination efficacy using highly standardized solution and surface tests was established and validated. All tested sodium hypochlorite-based reagents proved to be highly efficient in nucleic acid decontamination even after short reaction times. For DNA Away, a sodium hydroxide-based decontamination product, dose- and time-dependent effectiveness was ascertained. For two other commercial decontamination reagents, the phosphoric acid-based DNA Remover and the non-enzymatic reagent DNA-ExitusPlus™ IF, no reduction of amplifiable DNA/RNA was observed. In conclusion, a simple test procedure for evaluation of the elimination efficacy of decontamination reagents against amplifiable nucleic acid is presented.

  19. Biopolymers: protein and nucleic acids. Annual report, 15 September 1987-14 September 1988

    Energy Technology Data Exchange (ETDEWEB)

    Richards, J.H.; Abelson, J.N.; Dervan, P.B.; Hood, L.H.; Simon, M.I.

    1988-09-15

    The work focuses on learning the principles that govern interactions between proteins and nucleic acids, both DNA and RNA (specifically tRNA). With these principles as guides peptides (of about 50 amino acids) that bind to specific regions of DNA are being synthesized. Various reactive functionalities are being attached to the synthetic peptides to generate reagents that cleave DNA specifically at the site to which the peptide binds. The work also involves biophysical studies of the protein/nucleic acid complexes in order to expand our understanding of the principles of protein binding to nucleic acids. Development of improved procedures for the chemical synthesis of peptides forms another important aspect of the program.

  20. ABIOGENIC INFORMATION COUPLING BETWEEN NUCLEIC ACID AND PROTEIN,OR, HOW PROTEIN AND DNA WERE MARRIED

    Energy Technology Data Exchange (ETDEWEB)

    Calvin, Melvin

    1968-12-01

    There is now experimental evidence for selectivity between the amino acid and the nucleic acid base which is the beginning of the chemical translation process from one linear system to the other. The linear system of the nucleic acid is, of course, an excellent place to store the information, whereas the linear system of the polypeptide, on the other hand, is the versatile system which can perform many different types of reactions but is unable to store information reliably. The experiments the author has described here may represent the beginning of the method of coupling of those two essential qualities which are required for the generation and evolution of a living organism.

  1. Exonuclease III-boosted cascade reactions for ultrasensitive SERS detection of nucleic acids.

    Science.gov (United States)

    Sun, Yudie; Peng, Pai; Guo, Ruiyan; Wang, Huihui; Li, Tao

    2018-05-01

    A variety of nucleic acid amplification techniques have been integrated into different detection methods to promote the development of sensitive and convenient analysis of nucleic acids. However, it is still in urgent need to develop amplified nucleic acid biosensors for the analysis of susceptible gene and even distinguishing single-base mismatched DNA in complex biological samples. Benefiting from the achieved detection strategies, here we boost isothermal nucleic acid amplification by resorting to enzyme amplification, and combine this two-stage amplification method with surface-enhanced Raman spectroscopy (SERS) to develop a signal-on nucleic acid detection platform. Due to the high cleavage efficiency of Exonuclease III (Exo III), a large amount of trigger DNA are produced to initiate multiple hybridization chain reaction circles. The product structure tagged with Tamra is then anchored onto the plasmonic SERS substrate and meanwhile enriched. It is demonstrated that this detection platform is sensitive toward the myocardial infarction disease related gene. A detection limit of 1 fM for the gene analysis in a linear relationship in the wide range from 1 fM to 10nM is achieved, better than most of previous counterparts. Meanwhile, our developed detection platform exhibits a high selectivity for the target gene over mismatched analogues. Our strategy provides a robust tool for signal amplification of gene detection even in blood samples. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Coarse-Grained Modeling of Nucleic Acids Using Anisotropic Gay-Berne and Electric Multipole Potentials.

    Science.gov (United States)

    Li, Guohui; Shen, Hujun; Zhang, Dinglin; Li, Yan; Wang, Honglei

    2016-02-09

    In this work, we attempt to apply a coarse-grained (CG) model, which is based on anisotropic Gay-Berne and electric multipole (EMP) potentials, to the modeling of nucleic acids. First, a comparison has been made between the CG and atomistic models (AMBER point-charge model) in the modeling of DNA and RNA hairpin structures. The CG results have demonstrated a good quality in maintaining the nucleic acid hairpin structures, in reproducing the dynamics of backbone atoms of nucleic acids, and in describing the hydrogen-bonding interactions between nucleic acid base pairs. Second, the CG and atomistic AMBER models yield comparable results in modeling double-stranded DNA and RNA molecules. It is encouraging that our CG model is capable of reproducing many elastic features of nucleic acid base pairs in terms of the distributions of the interbase pair step parameters (such as shift, slide, tilt, and twist) and the intrabase pair parameters (such as buckle, propeller, shear, and stretch). Finally, The GBEMP model has shown a promising ability to predict the melting temperatures of DNA duplexes with different lengths.

  3. LINE-1 retrotransposition requires the nucleic acid chaperone activity of the ORF1 protein.

    Science.gov (United States)

    Martin, Sandra L; Cruceanu, Margareta; Branciforte, Dan; Wai-Lun Li, Patrick; Kwok, Stanley C; Hodges, Robert S; Williams, Mark C

    2005-05-06

    LINE-1 is a highly successful, non-LTR retrotransposon that has played a leading role in shaping mammalian genomes. These elements move autonomously through an RNA intermediate using target-primed reverse transcription (TPRT). L1 encodes two essential polypeptides for retrotransposition, the products of its two open reading frames, ORF1 and ORF2. The exact function of the ORF1 protein (ORF1p) in L1 retrotransposition is unknown, although it is an RNA-binding protein that can act as a nucleic acid chaperone. Here, we investigate the requirements for these two activities in L1 retrotransposition by examining the consequences of mutating two adjacent and highly conserved arginine residues in the ORF1p from mouse L1. Substitution of both arginine residues with alanine strongly reduces the affinity of the protein for single-stranded nucleic acid, whereas substitution of one or both with lysine has only minimal effects on this feature. Rather, the lysine substitutions alter the delicate balance between the ORF1 protein's melting and reannealing activities, thereby reducing its nucleic acid chaperone activity. These findings establish the importance of the nucleic acid chaperone activity of ORF1p to successful L1 retrotransposition, and provide insight into the essential properties of nucleic acid chaperones.

  4. [Study on the feasibility of human papilloma virus subtypes detection by automatic nucleic acid extraction workstation].

    Science.gov (United States)

    Zhang, Rui; Ye, Ali; Dou, Yaling; Gan, Yong; Kong, Lingjun; Chen, Yu; Xu, Yingchun

    2015-12-08

    To verify the feasibility of human papilloma virus(HPV) subtypes detection by AUTRAX automatic nucleic acid extraction workstation. A total of 183 HPV test samples (2 562 types) were collected during August 2014 in Peking Union Medical College Hospital. Nucleic acid determination kit of high-risk HPV types (16, 18, 35, 39, 58, 31, 33, 68, 56, 45, 59, 51, 52) and 6 , 11 type (real-time PCR) were applied for detection. Each sample was divided into two parts. One part was treated with manual extraction, which entailed manually preparing PCR reaction system and added the sample to the PCR plate. Another was treated with the AUTRAX automatic nucleic acid extraction workstation, which automatically prepared the reaction system and added to the PCR board. These two parts proceeded to the real-time PCR detection. The result of manual extraction was set as the golden standard and the Kappa consistency analysis was conducted. Meanwhile, precision and pollution prevention of the AUTRAX were verified. The sensitivity of AUTRAX automatic nucleic acids extraction workstation was 97.12% (101/104). The specificity was 99.51% (2 446/2 458). The accuracy was 99.42% (2 547/2 562). The result of Kappa consistency analysis showed that the two parts were the same (Kappa=0.926). Coefficient of variation (CV) of each HPV types was less than 5%.No pollution phenomenon was found. AUTRAX automatic nucleic acids extraction workstation can be used for the HPV subtypes detection in clinical settings.

  5. [Microbial bioavailability of dissolved nucleic acids across the estuarine salinity gradient].

    Science.gov (United States)

    Yang, Qing-Qing; Li, Peng-Hui; Huang, Qing-Hui

    2013-07-01

    As an important component of dissolved organic matter (DOM), nucleic acids (DNA and RNA) are essential nutrient and energy sources in aquatic microbial food web. Therefore, it is important to understand the bioavailability of nucleic acids. The bioavailability of nucleic acids was investigated by a batch of incubation experiments, adding fish DNA and yeast RNA into water samples with different salinity collected from the Yangtze River estuary in the spring of 2012. According to the results, 20%-50% of dissolved DNA was transformed into particulate DNA quickly with the conversion rates increasing with salinity, only 10% dissolved RNA was transformed into particulate RNA and the salinity had no effect on the conversation rates. In each incubation experiment, the microbial utilization kinetic curves of dissolved nucleic acids were fitted to the Sigmoid model. There were lag periods of 30-80 hours followed by the rapid utilization phase and then the stagnation phase. The results also showed that the bacteria in seawater had higher maximum utilization rate than the bacteria in estuarine and fresh water. Dissolved nucleic acids spiked in estuarine water can be bound to colloids and particles at some extent, only those free dissolved or enzymatically hydrolysable forms are bioavailable. The percentage of bioavailable RNA (80%-90%) was significantly higher than that of bioavailable DNA and it did not change significantly with salinity while the percent of bioavailable DNA decreased from 78% to 50% with salinity. Therefore, the speciation and bioavailability are significantly different between DNA and RNA across the estuarine salinity gradient.

  6. Phytoagents for Cancer Management: Regulation of Nucleic Acid Oxidation, ROS, and Related Mechanisms

    Directory of Open Access Journals (Sweden)

    Wai-Leng Lee

    2013-01-01

    Full Text Available Accumulation of oxidized nucleic acids causes genomic instability leading to senescence, apoptosis, and tumorigenesis. Phytoagents are known to reduce the risk of cancer development; whether such effects are through regulating the extent of nucleic acid oxidation remains unclear. Here, we outlined the role of reactive oxygen species in nucleic acid oxidation as a driving force in cancer progression. The consequential relationship between genome instability and cancer progression highlights the importance of modulation of cellular redox level in cancer management. Current epidemiological and experimental evidence demonstrate the effects and modes of action of phytoagents in nucleic acid oxidation and provide rationales for the use of phytoagents as chemopreventive or therapeutic agents. Vitamins and various phytoagents antagonize carcinogen-triggered oxidative stress by scavenging free radicals and/or activating endogenous defence systems such as Nrf2-regulated antioxidant genes or pathways. Moreover, metal ion chelation by phytoagents helps to attenuate oxidative DNA damage caused by transition metal ions. Besides, the prooxidant effects of some phytoagents pose selective cytotoxicity on cancer cells and shed light on a new strategy of cancer therapy. The “double-edged sword” role of phytoagents as redox regulators in nucleic acid oxidation and their possible roles in cancer prevention or therapy are discussed in this review.

  7. [Genotoxic modification of nucleic acid bases and biological consequences of it. Review and prospects of experimental and computational investigations

    Science.gov (United States)

    Poltev, V. I.; Bruskov, V. I.; Shuliupina, N. V.; Rein, R.; Shibata, M.; Ornstein, R.; Miller, J.

    1993-01-01

    The review is presented of experimental and computational data on the influence of genotoxic modification of bases (deamination, alkylation, oxidation) on the structure and biological functioning of nucleic acids. Pathways are discussed for the influence of modification on coding properties of bases, on possible errors of nucleic acid biosynthesis, and on configurations of nucleotide mispairs. The atomic structure of nucleic acid fragments with modified bases and the role of base damages in mutagenesis and carcinogenesis are considered.

  8. Lateral flow microarrays: a novel platform for rapid nucleic acid detection based on miniaturized lateral flow chromatography

    OpenAIRE

    Carter, Darren J.; Cary, R. Bruce

    2007-01-01

    Widely used nucleic acid assays are poorly suited for field deployment where access to laboratory instrumentation is limited or unavailable. The need for field deployable nucleic acid detection demands inexpensive, facile systems without sacrificing information capacity or sensitivity. Here we describe a novel microarray platform capable of rapid, sensitive nucleic acid detection without specialized instrumentation. The approach is based on a miniaturized lateral flow device that makes use of...

  9. Synthesis of reactive nucleic acid analogues and their application for the study of structure and functions of biopolymers

    Energy Technology Data Exchange (ETDEWEB)

    Kanevskii, Igor' E; Kuznetsova, Svetlana A [Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow (Russian Federation)

    1998-07-31

    Data on the synthesis of reactive derivatives of nucleic acid analogues and their application for the study of structure and functions of biopolymers are generalised. The main types of such analogues including photoactivated reagents containing azidoaryl, halogeno, and thiol groups, psoralen and its derivatives, platinum-based reagents, and nucleic acid analogues containing substituted pyrophosphate or acyl phosphate internucleotide groups are presented. The mechanisms of interaction of these compounds with proteins and nucleic acids are considered. The prospects for the in vivo application of reactive nucleic acids in various systems are discussed. The bibliography includes 76 references.

  10. The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections.

    Science.gov (United States)

    Gray, J; Coupland, L J

    2014-01-01

    On 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG® Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene - in-vitro diagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

  11. Nucleic acid-based methods for the early detection of sepsis in heart transplant recipients

    Directory of Open Access Journals (Sweden)

    Sarvesh Pal Singh

    2016-01-01

    Full Text Available Nucleic acid-based tests (NABTs were developed to decrease the time required to identify microorganism in the pathological specimens. The commercially available NABTs are of two types – one those can be applied to positive cultures grown in blood culture bottles and other those can be applied directly to blood samples. The latter tests are polymerase chain reaction (PCR-based assays which can amplify the existing load of microorganism nucleic acids many a times like a culture in a blood culture bottle. Both tests then identify the pathogenic organisms with the use of specific nucleic acid probes. These tests have proven useful in management of heart transplant sepsis management.

  12. Novel fluorescent nanoparticles for ultrasensitive identification of nucleic acids by optical methods

    DEFF Research Database (Denmark)

    Mulberg, Mads Westergaard; Taskova, Maria; Thomsen, Rasmus P.

    2017-01-01

    For decades, the detection of nucleic acids and their interactions at low abundances has been a challenging task. Present nucleic acid diagnostics are primarily based on enzymatic reactions including sequencing, polymerase-chain reaction and microarrays. However, the use of enzymatic amplification...... interferes with the initial biomolecular system, is limited to in vitro assays, often time consuming and rather expensive. Therefore, there is interest in new amplification-free detection methods. A tremendous progress has been made in fluorescence based optical detection of biomolecules. In this work, we...... aimed at developing efficient tools for amplification-free nucleic acid detection. The result of simple and inexpensive polymerization in the presence of fluorescent dyes and additional functionalization reagents was ultra-bright fluorescent nanoparticles modified with additional groups...

  13. Templated synthesis of peptide nucleic acids via sequence-selective base-filling reactions.

    Science.gov (United States)

    Heemstra, Jennifer M; Liu, David R

    2009-08-19

    The templated synthesis of nucleic acids has previously been achieved through the backbone ligation of preformed nucleotide monomers or oligomers. In contrast, here we demonstrate templated nucleic acid synthesis using a base-filling approach in which individual bases are added to abasic sites of a peptide nucleic acid (PNA). Because nucleobase substrates in this approach are not self-reactive, a base-filling approach may reduce the formation of nontemplated reaction products. Using either reductive amination or amine acylation chemistries, we observed efficient and selective addition of each of the four nucleobases to an abasic site in the middle of the PNA strand. We also describe the addition of single nucleobases to the end of a PNA strand through base filling, as well as the tandem addition of two bases to the middle of the PNA strand. These findings represent an experimental foundation for nonenzymatic information transfer through base filling.

  14. Nucleic acids in the last stages of maturation of two varieties

    Directory of Open Access Journals (Sweden)

    R. Górecki

    2015-01-01

    Full Text Available Using the MAK column chromatography method the process of wheat grain maturation was found to be closely correlated with nucleic acid metabolism. The amount of nucleic acids in the embryos increased until the end of the maturation of the grain. This increase was the result of an intensive synthesis mainly of rRNA fractions and to some extent also of sRNA. The quantitative proportion of various fractions of nucleic acids also changed in the last stages of grain maturation. In the period of wax maturity in wheat embryos a higher content of 4S and 5S RNA was found which partly explains a higher viability of these seeds.

  15. Label-free functional nucleic acid sensors for detecting target agents

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Yi; Xiang, Yu

    2015-01-13

    A general methodology to design label-free fluorescent functional nucleic acid sensors using a vacant site approach and an abasic site approach is described. In one example, a method for designing label-free fluorescent functional nucleic acid sensors (e.g., those that include a DNAzyme, aptamer or aptazyme) that have a tunable dynamic range through the introduction of an abasic site (e.g., dSpacer) or a vacant site into the functional nucleic acids. Also provided is a general method for designing label-free fluorescent aptamer sensors based on the regulation of malachite green (MG) fluorescence. A general method for designing label-free fluorescent catalytic and molecular beacons (CAMBs) is also provided. The methods demonstrated here can be used to design many other label-free fluorescent sensors to detect a wide range of analytes. Sensors and methods of using the disclosed sensors are also provided.

  16. Development of Chemiluminescent Lateral Flow Assay for the Detection of Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Sam R. Nugen

    2012-01-01

    Full Text Available Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety. Herein we report the development of a nucleic acid sequence-based lateral flow assay which achieves a low limit of detection using chemiluminescence. On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level. To demonstrate this assay, we detected synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, with relevance in human and animal health in sub-Saharan Africa. The intensity of the chemiluminescent signal was evaluated by using a charge-coupled device as well as a microtiter plate reader. We demonstrated that our lateral flow chemiluminescent assay has a very low limit of detection and is easy to use. The limit of detection was determined to be 0.5 fmols of nucleic acid target.

  17. Development of Chemiluminescent Lateral Flow Assay for the Detection of Nucleic Acids

    Science.gov (United States)

    Wang, Yuhong; Fill, Catherine; Nugen, Sam R.

    2012-01-01

    Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety. Herein we report the development of a nucleic acid sequence-based lateral flow assay which achieves a low limit of detection using chemiluminescence. On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level. To demonstrate this assay, we detected synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, with relevance in human and animal health in sub-Saharan Africa. The intensity of the chemiluminescent signal was evaluated by using a charge-coupled device as well as a microtiter plate reader. We demonstrated that our lateral flow chemiluminescent assay has a very low limit of detection and is easy to use. The limit of detection was determined to be 0.5 fmols of nucleic acid target. PMID:25585630

  18. [Comparison of two nucleic acid extraction methods for norovirus in oysters].

    Science.gov (United States)

    Yuan, Qiao; Li, Hui; Deng, Xiaoling; Mo, Yanling; Fang, Ling; Ke, Changwen

    2013-04-01

    To explore a convenient and effective method for norovirus nucleic acid extraction from oysters suitable for long-term viral surveillance. Two methods, namely method A (glycine washing and polyethylene glycol precipitation of the virus followed by silica gel centrifugal column) and method B (protease K digestion followed by application of paramagnetic silicon) were compared for their performance in norovirus nucleic acid extraction from oysters. Real-time RT-PCR was used to detect norovirus in naturally infected oysters and in oysters with induced infection. The two methods yielded comparable positive detection rates for the samples, but the recovery rate of the virus was higher with method B than with method A. Method B is a more convenient and rapid method for norovirus nucleic acid extraction from oysters and suitable for long-term surveillance of norovirus.

  19. Probing nucleic acid-ion interactions with buffer exchange-atomic emission spectroscopy.

    Science.gov (United States)

    Greenfeld, Max; Herschlag, Daniel

    2009-01-01

    The ion atmosphere of nucleic acids directly affects measured biochemical and biophysical properties. However, study of the ion atmosphere is difficult due to its diffuse and dynamic nature. Standard techniques available have significant limitations in sensitivity, specificity, and directness of the assays. Buffer exchange-atomic emission spectroscopy (BE-AES) was developed to overcome many of the limitations of previously available techniques. This technique can provide a complete accounting of all ions constituting the ionic atmosphere of a nucleic acid at thermodynamic equilibrium. Although initially developed for the study of the ion atmosphere of nucleic acids, BE-AES has also been applied to study site-bound ions in RNA and protein. Copyright © 2009 Elsevier Inc. All rights reserved.

  20. Engineering nucleic acid structures for programmable molecular circuitry and intracellular biocomputation

    Science.gov (United States)

    Li, Jiang; Green, Alexander A.; Yan, Hao; Fan, Chunhai

    2017-11-01

    Nucleic acids have attracted widespread attention due to the simplicity with which they can be designed to form discrete structures and programmed to perform specific functions at the nanoscale. The advantages of DNA/RNA nanotechnology offer numerous opportunities for in-cell and in-vivo applications, and the technology holds great promise to advance the growing field of synthetic biology. Many elegant examples have revealed the potential in integrating nucleic acid nanostructures in cells and in vivo where they can perform important physiological functions. In this Review, we summarize the current abilities of DNA/RNA nanotechnology to realize applications in live cells and then discuss the key problems that must be solved to fully exploit the useful properties of nanostructures. Finally, we provide viewpoints on how to integrate the tools provided by DNA/RNA nanotechnology and related new technologies to construct nucleic acid nanostructure-based molecular circuitry for synthetic biology.

  1. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time.

    Science.gov (United States)

    Gandelman, Olga A; Church, Vicki L; Moore, Cathy A; Kiddle, Guy; Carne, Christopher A; Parmar, Surendra; Jalal, Hamid; Tisi, Laurence C; Murray, James A H

    2010-11-30

    The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal

  2. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time.

    Directory of Open Access Journals (Sweden)

    Olga A Gandelman

    Full Text Available BACKGROUND: The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. PRINCIPAL FINDINGS: Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. CONCLUSIONS: The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a

  3. A nucleotide-independent cyclic nitroxide label for monitoring segmental motions in nucleic acids.

    Science.gov (United States)

    Nguyen, Phuong H; Popova, Anna M; Hideg, Kálmán; Qin, Peter Z

    2015-01-01

    Spin labels, which are chemically stable radicals attached at specific sites of a bio-molecule, enable investigations on structure and dynamics of proteins and nucleic acids using techniques such as site-directed spin labeling and paramagnetic NMR. Among spin labels developed, the class of rigid labels have limited or no independent motions between the radical bearing moiety and the target, and afford a number of advantages in measuring distances and monitoring local dynamics within the parent bio-molecule. However, a general method for attaching a rigid label to nucleic acids in a nucleotide-independent manner has not been reported. We developed an approach for installing a nearly rigid nitroxide spin label, designated as R5c, at a specific site of the nucleic acid backbone in a nucleotide-independent manner. The method uses a post-synthesis approach to covalently attach the nitroxide moiety in a cyclic fashion to phosphorothioate groups introduced at two consecutive nucleotides of the target strand. R5c-labeled nucleic acids are capable of pairing with their respective complementary strands, and the cyclic nature of R5c attachment significantly reduced independence motions of the label with respect to the parent duplex, although it may cause distortion of the local environment at the site of labeling. R5c yields enhanced sensitivity to the collective motions of the duplex, as demonstrated by its capability to reveal changes in collective motions of the substrate recognition duplex of the 120-kDa Tetrahymena group I ribozyme, which elude detection by a flexible label. The cyclic R5c nitroxide can be efficiently attached to a target nucleic acid site using a post-synthetic coupling approach conducted under mild biochemical conditions, and serves as a viable label for experimental investigation of segmental motions in nucleic acids, including large folded RNAs.

  4. Disposable nucleic acid biosensors based on gold nanoparticle probes and lateral flow strip.

    Science.gov (United States)

    Mao, Xun; Ma, Yunqing; Zhang, Aiguo; Zhang, Lurong; Zeng, Lingwen; Liu, Guodong

    2009-02-15

    In this article, we describe a disposable nucleic acid biosensor (DNAB) for low-cost and sensitive detection of nucleic acid samples in 15 min. Combining the unique optical properties of gold nanoparticles (Au-NP) and the high efficiency of chromatographic separation, sandwich-type DNA hybridization reactions were realized on the lateral flow strips, which avoid multiple incubation, separation, and washing steps in the conventional nucleic acid biosensors. The captured Au-NP probes on the test zone and control zone of the biosensor produced the characteristic red bands, enabling visual detection of nucleic acid samples without instrumentation. The quantitative detection was performed by reading the intensities of the produced red bands with a portable strip reader. The parameters (e.g., the concentration of reporter probe, the size of Au-NP, the amount of Au-NP-DNA probe, lateral flow membranes, and the concentration of running buffer) that govern the sensitivity and reproducibility of the sensor were optimized. The response of the optimized device is highly linear over the range of 1-100 nM target DNA, and the limit of detection is estimated to be 0.5 nM in association with a 15 min assay time. The sensitivity of the biosensor was further enhanced by using horseradish peroxidase (HRP)-Au-NP dual labels which ensure a quite low detection limit of 50 pM. The DNAB has been applied for the detection of human genomic DNA directly with a detection limit of 2.5 microg/mL (1.25 fM) by adopting well-designed DNA probes. The new nucleic acid biosensor thus provides a rapid, sensitive, low cost, and quantitative tool for the detection of nucleic acid samples. It shows great promise for in-field and point-of-care diagnosis of genetic diseases and detection of infectious agents or warning against biowarfare agents.

  5. Nonspecific prion protein-nucleic acid interactions lead to different aggregates and cytotoxic species.

    Science.gov (United States)

    Macedo, Bruno; Millen, Thiago A; Braga, Carolina A C A; Gomes, Mariana P B; Ferreira, Priscila S; Kraineva, Julia; Winter, Roland; Silva, Jerson L; Cordeiro, Yraima

    2012-07-10

    A misfolded form of the prion protein (PrP) is the primary culprit in mammalian prion diseases. It has been shown that nucleic acids catalyze the misfolding of cellular PrP into a scrapie-like conformer. It has also been observed that the interaction of PrP with nucleic acids is nonspecific and that the complex can be toxic to cultured cells. No direct correlation has yet been drawn between changes in PrP structure and toxicity due to nucleic acid binding. Here we asked whether different aggregation, stability, and toxicity effects are detected when nonrelated DNA sequences interact with recombinant PrP. Using spectroscopic techniques to analyze PrP tertiary and secondary structure and cellular assays to assess toxicity, we found that rPrP-DNA interactions lead to different aggregated species, depending on the sequence and size of the oligonucleotide tested. A 21-mer DNA sequence (D67) induced higher levels of aggregation and also dissimilar structural changes in rPrP, compared to binding to oligonucleotides with the same length and different nucleotide sequences or different GC contents. The rPrP-D67 complex induced significant cell dysfunction, which appears to be correlated with the biophysical properties of the complex. Although sequence specificity is not apparent for PrP-nucleic acid interactions, we believe that particular nucleic acid patterns, possibly related to GC content, oligonucleotide length, and structure, govern PrP recognition. Understanding the structural and cellular effects observed for PrP-nucleic acid complexes may shed light on the still mysterious pathology of the prion protein.

  6. Interactions of hydrated divalent metal cations with nucleic acid bases. How to relate the gas phase data to solution situation and binding selectivity in nucleic acids

    Czech Academy of Sciences Publication Activity Database

    Šponer, Judit E.; Sychrovský, Vladimír; Hobza, Pavel; Šponer, Jiří

    2004-01-01

    Roč. 6, č. 10 (2004), s. 2772-2780 ISSN 1463-9076 R&D Projects: GA MŠk LN00A016; GA MŠk LN00A032 Grant - others:Wellcome Trust(GB) GR067507MF Institutional research plan: CEZ:AV0Z5004920 Keywords : nucleic acids * gas phase * guanine Subject RIV: BO - Biophysics Impact factor: 2.076, year: 2004

  7. JAWS: Just Add Water System - A device for detection of nucleic acids in Martian ice caps

    DEFF Research Database (Denmark)

    Hansen, Anders J.; Willerslev, Eske; Mørk, Søren

    2002-01-01

    The design of a device for nucleic acid detection in the Martian ice caps is presented; the Just Add Water System (JAWS). It is based on fiber-optic PNA (peptide nucleic acid) light up probe random microsphere universal array technology. JAWS is designed to be part of a larger system...... with a regulation of pH and salt concentrations e.g. the MOD systems and could be installed on a planetary probe melting its way down the Martian ice caps e.g. the NASA Cryobot. JAWS can be used for detection of remains of ancient life preserved in the Martian ice as well as for detection of contamination brought...

  8. Efficient Reverse Transcription Using Locked Nucleic Acid Nucleotides towards the Evolution of Nuclease Resistant RNA Aptamers

    DEFF Research Database (Denmark)

    Crouzier, Lucile; Dubois, Camille; Edwards, Stacey L

    2012-01-01

    Modified nucleotides are increasingly being utilized in the de novo selection of aptamers for enhancing their drug-like character and abolishing the need for time consuming trial-and-error based post-selection modifications. Locked nucleic acid (LNA) is one of the most prominent and successful...... nucleic acid analogues because of its remarkable properties, and widely explored as building blocks in therapeutic oligonucleotides. Evolution of LNA-modified RNA aptamers requires an efficient reverse transcription method for PCR enrichment of the selected RNA aptamer candidates. Establishing this key...... step is a pre-requisite for performing LNA-modified RNA aptamer selection....

  9. The predictive power of synthetic nucleic acid technologies in RNA biology.

    Science.gov (United States)

    Chakraborty, Saikat; Mehtab, Shabana; Krishnan, Yamuna

    2014-06-17

    CONSPECTUS: The impact of nucleic acid nanotechnology in terms of transforming motifs from biology in synthetic and translational ways is widely appreciated. But it is also emerging that the thinking and vision behind nucleic acids as construction material has broader implications, not just in nanotechnology or even synthetic biology, but can feed back into our understanding of biology itself. Physicists have treated nucleic acids as polymers and connected physical principles to biology by abstracting out the molecular interactions. In contrast, biologists delineate molecular players and pathways related to nucleic acids and how they may be networked. But in vitro nucleic acid nanotechnology has provided a valuable framework for nucleic acids by connecting its biomolecular interactions with its materials properties and thereby superarchitecture ultramanipulation that on multiple occasions has pre-empted the elucidation of how living cells themselves are exploiting these same structural concepts. This Account seeks to showcase the larger implications of certain architectural principles that have arisen from the field of structural DNA/RNA nanotechnology in biology. Here we draw connections between these principles and particular molecular phenomena within living systems that have fed in to our understanding of how the cell uses nucleic acids as construction material to achieve different functions. We illustrate this by considering a few exciting and emerging examples in biology in the context of both switchable systems and scaffolding type systems. Due to the scope of this Account, we will focus our discussion on examples of the RNA scaffold as summarized. In the context of switchable RNA architectures, the synthetic demonstration of small molecules blocking RNA translation preceded the discovery of riboswitches. In another example, it was after the description of aptazymes that the first allosteric ribozyme, glmS, was discovered. In the context of RNA architectures

  10. Nanomaterials for delivery of nucleic acid to the central nervous system (CNS)

    DEFF Research Database (Denmark)

    Wang, Danyang; Wu, Lin-Ping

    2017-01-01

    -related disease, such as neurodegeneration and disorders, suitable, safe and effective drug delivery nanocarriers have to been developed to overcome the blood brain barrier (BBB), which is the most inflexible barrier in human body. Here, we highlight the structure and function of barriers in the central nervous......Billions of dollars have been invested in the therapeutic application of nucleic acid-based agents in humans in recent years. There are inspirable data from ongoing clinical trial for different diseases. However, in order to widely apply nucleic acid in prevention, diagnosis and treatment of age...

  11. Sensitive detection of nucleic acids by PNA hybridization directed co-localization of fluorescent beads

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Deborggraeve, Stijn; Büscher, Philippe

    2011-01-01

    We have designed a pair of biotinylated peptide nucleic acid (PNA) probes targeting two sequences in 18S rRNA (from the parasite Trypanosoma brucei) at a distance of 191 nt (corresponding to maximum distance of ca. 60 nm) from each other. The PNA probes were individually bound to (strept)avidin-c......We have designed a pair of biotinylated peptide nucleic acid (PNA) probes targeting two sequences in 18S rRNA (from the parasite Trypanosoma brucei) at a distance of 191 nt (corresponding to maximum distance of ca. 60 nm) from each other. The PNA probes were individually bound to (strept...

  12. Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases.

    Science.gov (United States)

    Crouzier, Lucile; Dubois, Camille; Wengel, Jesper; Veedu, Rakesh N

    2012-07-15

    Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Accuracy assessment of the linear Poisson-Boltzmann equation and reparametrization of the OBC generalized Born model for nucleic acids and nucleic acid-protein complexes.

    Science.gov (United States)

    Fogolari, Federico; Corazza, Alessandra; Esposito, Gennaro

    2015-04-05

    The generalized Born model in the Onufriev, Bashford, and Case (Onufriev et al., Proteins: Struct Funct Genet 2004, 55, 383) implementation has emerged as one of the best compromises between accuracy and speed of computation. For simulations of nucleic acids, however, a number of issues should be addressed: (1) the generalized Born model is based on a linear model and the linearization of the reference Poisson-Boltmann equation may be questioned for highly charged systems as nucleic acids; (2) although much attention has been given to potentials, solvation forces could be much less sensitive to linearization than the potentials; and (3) the accuracy of the Onufriev-Bashford-Case (OBC) model for nucleic acids depends on fine tuning of parameters. Here, we show that the linearization of the Poisson Boltzmann equation has mild effects on computed forces, and that with optimal choice of the OBC model parameters, solvation forces, essential for molecular dynamics simulations, agree well with those computed using the reference Poisson-Boltzmann model. © 2015 Wiley Periodicals, Inc.

  14. New polymer of lactic-co-glycolic acid-modified polyethylenimine for nucleic acid delivery.

    Science.gov (United States)

    Lü, Jian-Ming; Liang, Zhengdong; Wang, Xiaoxiao; Gu, Jianhua; Yao, Qizhi; Chen, Changyi

    2016-08-01

    To develop an improved delivery system for nucleic acids. We designed, synthesized and characterized a new polymer of lactic-co-glycolic acid-modified polyethylenimine (LGA-PEI). Functions of LGA-PEI polymer were determined. The new LGA-PEI polymer spontaneously formed nanoparticles (NPs) with DNA or RNA, and showed higher DNA or RNA loading efficiency, higher or comparable transfection efficacy, and lower cytotoxicity in several cell types including PANC-1, Jurkat and HEK293 cells, when compared with lipofectamine 2000, branched or linear PEI (25 kDa). In nude mouse models, LGA-PEI showed higher delivery efficiency of plasmid DNA or miRNA mimic into pancreatic and ovarian xenograft tumors. LGA-PEI/DNA NPs showed much lower toxicity than control PEI NPs in mouse models. The new LGA-PEI polymer is a safer and more effective system to deliver DNA or RNA than PEI.

  15. Fe-nitrilotriacetic acid coordination polymer nanowires: an effective sensing platform for fluorescence-enhanced nucleic acid detection

    Science.gov (United States)

    Zhou, Yunchun; Liu, Qian; Sun, Xuping; Kong, Rongmei

    2017-02-01

    The determination of specific nucleic acid sequences is key in identifying disease-causing pathogens and genetic diseases. In this paper we report the utilization of Fe-nitrilotriacetic acid coordination polymer nanowires as an effective nanoquencher for fluorescence-enhanced nucleic acid detection. The detection is fast and the whole process can be completed within 15 min. This nanosensor shows a low detection limit of 0.2 nM with selectivity down to single-base mismatch. This work provides us with an attractive sensing platform for applications.

  16. Triazole linkages and backbone branches in nucleic acids for biological and extra-biological applications

    Science.gov (United States)

    Paredes, Eduardo

    The recently increasing evidence of nucleic acids' alternative roles in biology and potential as useful nanomaterials and therapeutic agents has enabled the development of useful probes, elaborate nanostructures and therapeutic effectors based on nucleic acids. The study of alternative nucleic acid structure and function, particularly RNA, hinges on the ability to introduce site-specific modifications that either provide clues to the nucleic acid structure function relationship or alter the nucleic acid's function. Although the available chemistries allow for the conjugation of useful labels and molecules, their limitations lie in their tedious conjugation conditions or the lability of the installed probes. The development and optimization of click chemistry with RNA now provides the access to a robust and orthogonal conjugation methodology while providing stable conjugates. Our ability to introduce click reactive groups enzymatically, rather than only in the solid-phase, allows for the modification of larger, more cell relevant RNAs. Additionally, ligation of modified RNAs with larger RNA constructs through click chemistry represents an improvement over traditional ligation techniques. We determined that the triazole linkage generated through click chemistry is compatible in diverse nucleic acid based biological systems. Click chemistry has also been developed for extra-biological applications, particularly with DNA. We have expanded its use to generate useful polymer-DNA conjugates which can form controllable soft nanoparticles which take advantage of DNA's properties, i.e. DNA hybridization and computing. Additionally, we have generated protein-DNA conjugates and assembled protein-polymer hybrids mediated by DNA hybridization. The use of click chemistry in these reactions allows for the facile synthesis of these unnatural conjugates. We have also developed backbone branched DNA through click chemistry and showed that these branched DNAs are useful in generating

  17. The evolution of bat nucleic acid-sensing Toll-like receptors.

    Science.gov (United States)

    Escalera-Zamudio, Marina; Zepeda-Mendoza, M Lisandra; Loza-Rubio, Elizabeth; Rojas-Anaya, Edith; Méndez-Ojeda, Maria L; Arias, Carlos F; Greenwood, Alex D

    2015-12-01

    We characterized the nucleic acid-sensing Toll-like receptors (TLR) of a New World bat species, the common vampire bat (Desmodus rotundus), and through a comparative molecular evolutionary approach searched for general adaptation patterns among the nucleic acid-sensing TLRs of eight different bats species belonging to three families (Pteropodidae, Vespertilionidae and Phyllostomidae). We found that the bat TLRs are evolving slowly and mostly under purifying selection and that the divergence pattern of such receptors is overall congruent with the species tree, consistent with the evolution of many other mammalian nuclear genes. However, the chiropteran TLRs exhibited unique mutations fixed in ligand-binding sites, some of which involved nonconservative amino acid changes and/or targets of positive selection. Such changes could potentially modify protein function and ligand-binding properties, as some changes were predicted to alter nucleic acid binding motifs in TLR 9. Moreover, evidence for episodic diversifying selection acting specifically upon the bat lineage and sublineages was detected. Thus, the long-term adaptation of chiropterans to a wide variety of environments and ecological niches with different pathogen profiles is likely to have shaped the evolution of the bat TLRs in an order-specific manner. The observed evolutionary patterns provide evidence for potential functional differences between bat and other mammalian TLRs in terms of resistance to specific pathogens or recognition of nucleic acids in general. © 2015 John Wiley & Sons Ltd.

  18. Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

    Science.gov (United States)

    Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A.

    2012-05-01

    Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

  19. Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

    Energy Technology Data Exchange (ETDEWEB)

    Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A., E-mail: alashkov83@gmail.com [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2012-05-15

    Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

  20. Viral Nucleic Acids in the Serum Are Dependent on Blood Sampling Site in Patients with Clinical Suspicion of Myocarditis.

    Science.gov (United States)

    Pawlak, Agnieszka; Przybylski, Maciej; Durlik, Marek; Gil, Katarzyna; Nasierowska-Guttmejer, Anna M; Byczkowska, Katarzyna; Ziemba, Andrzej; Gil, Robert J

    2016-01-01

    The meaning of viral nucleic acids in the myocardium in many cases is difficult for clinical interpretation, whereas the presence of viral nucleic acids in the serum is a marker of active infection. We determined the diagnostic value of viral nucleic acids in ventricular serum and peripheral serum samples in comparison with endomyocardial biopsy (EMB) specimens in patients with clinically suspected myocarditis. The viral nucleic acid evaluation was performed in serum samples and EMB specimens by real-time PCR in 70 patients (age: 47 ± 16 years). The biopsy specimens were examined by histo- and immunohistochemistry to detect inflammatory response. The viral nucleic acids were detected in ventricular and peripheral serum, and EMB samples of 10 (14%), 14 (20%), and 32 (46%) patients, respectively. Notably, viral nucleic acids of the same virus as in the EMB sample were present more often in ventricular than in peripheral serum (60 vs. 7%, p = 0.01). A significant concurrence was observed between the positive and the negative results of viral nucleic acids present in EMB and ventricular serum (p = 0.0001). The detection of the same viral nucleic acid type in the myocardium and in ventricular serum being significantly more frequent than in the peripheral serum may suggest that the site of the blood collection is important for more precise and reliable confirmation of the active viral replication in the heart. © 2017 S. Karger AG, Basel.

  1. 76 FR 72950 - Draft Guidance for Industry: Use of Nucleic Acid Tests on Pooled and Individual Samples From...

    Science.gov (United States)

    2011-11-28

    ... HUMAN SERVICES Food and Drug Administration Draft Guidance for Industry: Use of Nucleic Acid Tests on... document entitled ``Guidance for Industry: Use of Nucleic Acid Tests (NAT) on Pooled and Individual Samples... Whole Blood and blood components for transfusion or for further manufacture, including recovered plasma...

  2. 77 FR 68133 - Guidance for Industry: Use of Nucleic Acid Tests on Pooled and Individual Samples From Donors of...

    Science.gov (United States)

    2012-11-15

    ... HUMAN SERVICES Food and Drug Administration Guidance for Industry: Use of Nucleic Acid Tests on Pooled... a document entitled ``Guidance for Industry: Use of Nucleic Acid Tests on Pooled and Individual... and blood components for transfusion or for further manufacture, including recovered plasma, Source...

  3. The pattern recognition molecule deleted in malignant brain tumors 1 (DMBT1) and synthetic mimics inhibit liposomal nucleic acid delivery

    DEFF Research Database (Denmark)

    Lund Hansen, Pernille; Blaich, Stephanie; End, Caroline

    2011-01-01

    Liposomal nucleic acid delivery is a preferred option for therapeutic settings. The cellular pattern recognition molecule DMBT1, secreted at high levels in various diseases, and synthetic mimics efficiently inhibit liposomal nucleic acid delivery to human cells. These findings may have relevance ...

  4. DMPD: Nucleic acid-sensing TLRs as modifiers of autoimmunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available S. J Immunol. 2006 Nov 15;177(10):6573-8. (.png) (.svg) (.html) (.csml) Show Nucleic acid-sensing TLRs as mo...way - PNG File (.png) SVG File (.svg) HTML File (.html) CSML File (.csml) Open .csml file with CIOPlayer Ope

  5. Nucleic acids-based therapeutics in the battle against pathogenic viruses

    NARCIS (Netherlands)

    Haasnoot, Joost; Berkhout, Ben

    2009-01-01

    For almost three decades, researchers have studied the possibility to use nucleic acids as antiviral therapeutics. In theory, compounds such as antisense oligonucleotides, ribozymes, DNAzymes, and aptamers can be designed to trigger the sequence-specific inhibition of particular mRNA transcripts,

  6. Rapid identification of Stenotrophomonas maltophilia by peptide nucleic acid fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Knudsen, Nanna Reumert; Rasmussen, A. K. I.; Fiandaca, M. J.

    2014-01-01

    The objective of this study was to develop a novel peptide nucleic acid (PNA) probe for Stenotrophomonas maltophilia identification by fluorescence in situ hybridization (FISH). The probe was evaluated using 33 human and veterinary clinical S. maltophilia isolates and 45 reference strains...... horses. Altogether the study shows that this species-specific PNA FISH probe facilitates rapid detection of S. maltophilia in biological specimens....

  7. Survivin mRNA antagonists using locked nucleic acid, potential for molecular cancer therapy

    DEFF Research Database (Denmark)

    Fisker, Niels; Westergaard, Majken; Hansen, Henrik Frydenlund

    2007-01-01

    We have investigated the effects of different locked nucleic acid modified antisense mRNA antagonists against Survivin in a prostate cancer model. These mRNA antagonists were found to be potent inhibitors of Survivin expression at low nanomolar concentrations. Additionally there was a pronounced ...

  8. Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout.

    Science.gov (United States)

    Kong, Janay E; Wei, Qingshan; Tseng, Derek; Zhang, Jingzi; Pan, Eric; Lewinski, Michael; Garner, Omai B; Ozcan, Aydogan; Di Carlo, Dino

    2017-03-28

    Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/μL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

  9. Robert Feulgen Prize Lecture 1995. New approaches to in situ detection of nucleic acids.

    Science.gov (United States)

    Thiry, M

    1995-08-01

    The present paper reviews recent results obtained by different molecular biology-based, immunocytological approaches to the localization and identification of nucleic acids in sections of biological material. Examples of sensitive, high-resolution detection methods for RNA, DNA or specialized DNA regions are presented. Special emphasis is placed on the potential values and limitations of these new methods.

  10. Single-molecule pull-down for investigating protein–nucleic acid interactions

    NARCIS (Netherlands)

    Fareh, M.; Loeff, L.; Szczepaniak, M.; Haagsma, Anna C.; Yeom, K.H.; Joo, C.

    2016-01-01

    The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein

  11. Nucleic Acids Bind to Nanoparticulate iron (II) Monosulphide in Aqueous Solutions

    Science.gov (United States)

    Hatton, Bryan; Rickard, David

    2008-06-01

    In the hydrothermal FeS-world origin of life scenarios nucleic acids are suggested to bind to iron (II) monosulphide precipitated from the reaction between hydrothermal sulphidic vent solutions and iron-bearing oceanic water. In lower temperature systems, the first precipitate from this process is nanoparticulate, metastable FeSm with a mackinawite structure. Although the interactions between bulk crystalline iron sulphide minerals and nucleic acids have been reported, their reaction with nanoparticulate FeSm has not previously been investigated. We investigated the binding of different nucleic acids, and their constituents, to freshly precipitated, nanoparticulate FeSm. The degree to which the organic molecules interacted with FeSm is chromosomal DNA > RNA > oligomeric DNA > deoxadenosine monophosphate ≈ deoxyadenosine ≈ adenine. Although we found that FeSm does not fluoresce within the visible spectrum and there is no quantum confinement effect seen in the absorption, the mechanism of linkage of the FeSm to these biomolecules appears to be primarily electrostatic and similar to that found for the attachment of ZnS quantum dots. The results of a preliminary study of similar reactions with nanoparticulate CuS further supported the suggestion that the interaction mechanism was generic for nanoparticulate transition metal sulphides. In terms of the FeS-world hypothesis, the results of this study further support the idea that sulphide minerals precipitated at hydrothermal vents interact with biomolecules and could have assisted in the formation and polymerisation of nucleic acids.

  12. Nucleic acid and protein extraction from electropermeabilized E. coli cells on a microfluidic chip

    DEFF Research Database (Denmark)

    Matos, T.; Senkbeil, Silja; Mendonça, A.

    2013-01-01

    technique has been developed which is based on exposing E. coli cells to low voltages to allow extraction of nucleic acids and proteins. The flow-through electropermeability chip used consists of a microfluidic channel with integrated gold electrodes that promote cell envelope channel formation at low...

  13. Modulation of CpG oligodeoxynucleotide-mediated immune stimulation by locked nucleic acid (LNA)

    DEFF Research Database (Denmark)

    Vollmer, Jörg; Jepsen, Jan Stenvang; Uhlmann, Eugen

    2004-01-01

    Locked nucleic acid (LNA) is an RNA derivative that when introduced into oligodeoxynucleotides (ODN), mediates high efficacy and stability. CpG ODNs are potent immune stimulators and are recognized by toll-like receptor-9 (TLR9). Some phosphorothioate antisense ODNs bearing CpG dinucleotides have...

  14. Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR

    DEFF Research Database (Denmark)

    Hummelshoj, Lone; Ryder, Lars P; Madsen, Hans O

    2005-01-01

    Locked nucleic acid (LNA) is a modified DNA with increased binding affinityfor complementary DNA sequences. Our strategy was to use this property of LNA to inhibit undesired PCR amplification (e.g.,from contaminating genomic DNA) in a cDNA-based assay. By placing a short complementary LNA sequence...

  15. Recent Advances in Nucleic Acid-Based Delivery: From Bench to Clinical Trials in Genetic Diseases.

    Science.gov (United States)

    Oliveira, Cláudia; Ribeiro, António J; Veiga, Francisco; Silveira, Isabel

    2016-05-01

    Delivery of nucleic acids is the most promising therapy for many diseases that remain untreatable. Therefore, many research efforts have been put on finding a safe and efficient delivery system able to provide a sustained response. Viral vectors have proved to be the most efficient for delivery of nucleic acids and, thus, stand as the foremost vector used in current clinical trials. However, safety issues arise as a main concern and mitigate their use, impelling the improvement of non-viral alternatives. This review focuses on the recent advances in pre-clinical development of non-viral polyplexes and lipoplexes for nucleic acid-based delivery, in contrast with vectors being used in present clinical trials. Nucleic acid vectors for neurodegenerative ataxias, Parkinson's disease, retinitis pigmentosa, cystic fibrosis, hemophilia, pancreatic and lung cancer, and rheumatoid arthritis are discussed to illustrate current state of pre-clinical and clinical studies. Thereby, denoting the prospects for treatment of genetic diseases and elucidating the trend in non-viral vector development and improvement which is expected to significantly increase disease rescue exceeding the modest clinical successes observed so far.

  16. Stepping towards highly flexible aptamers: enzymatic recognition studies of unlocked nucleic acid nucleotides

    DEFF Research Database (Denmark)

    Dubois, Camille; Campbell, Meghan A; Edwards, Stacey L

    2012-01-01

    Enzymatic recognition of unlocked nucleic acid (UNA) nucleotides was successfully accomplished. Therminator DNA polymerase was found to be an efficient enzyme in primer extension reactions. Polymerase chain reaction (PCR) amplification of a 81 mer UNA-modified DNA library was efficiently achieved...

  17. Nucleic Acid Lateral Flow Immunoassay for the Detection of Pathogenic Bacteria from Food

    NARCIS (Netherlands)

    Blazkova, M.; Koets, M.; Wichers, J.H.; Amerongen, van A.; Fukal, L.; Rauch, P.

    2009-01-01

    Nucleic acid lateral flow immunoassay (NALFIA) is a method combining molecular biological principle of detection with immunochemical principle of visualisation. Following isolation of DNA from the sample, a duplex PCR with two primer sets, of which one was labelled with biotin and the other with

  18. Urinary markers of nucleic acid oxidation and cancer in type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Kasper Broedbaek

    2015-04-01

    Conclusions: Urinary excretion of the nucleic acid oxidation markers 8-oxodG and 8-oxoGuo at the time of diagnosis was not associated with cancer overall in type 2 diabetes patients. For site-specific cancers, risk elevations were seen for breast cancer (8-oxodG. These findings should be examined in future and larger studies.

  19. Response of sedimentary nucleic acids to benthic disturbance in the Central Indian Basin

    Digital Repository Service at National Institute of Oceanography (India)

    Fernandes, C.E.G.; DeSouza, M.J.B.D.; Nair, S.; LokaBharathi, P.A.

    Information on the response of nucleic acids (i.e., DNA and RNA) to simulated benthic disturbance was obtained from samples collected from eight sediment cores (0-10 cm) located in the Central Indian Basin (CIB). In general the total sedimentary DNA...

  20. Removal of contaminating DNA from commercial nucleic acid extraction kit reagents

    NARCIS (Netherlands)

    Mohammadi, Tamimount; Reesink, Henk W.; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.

    2005-01-01

    Due to contamination of DNA extraction reagents, false-positive results can occur when applying broad-range real-time PCR based on bacterial 16S rDNA. Filtration of the nucleic acid extraction kit reagents with GenElute Maxiprep binding columns was effective in removing this reagent-derived

  1. A chemical perspective on transcriptional fidelity dominant contributions of sugar integrity revealed by unlocked nucleic acids

    DEFF Research Database (Denmark)

    Xu, Liang; Plouffe, Steven W; Chong, Jenny

    2013-01-01

    Transcription unlocked: A synthetic chemical biology approach involving unlocked nucleic acids was used to dissect the contribution of sugar backbone integrity to the RNA Polymerase II (Pol II) transcription process. An unexpected dominant role for sugar-ring integrity in Pol II transcriptional...

  2. Facile functionalization of peptide nucleic acids (PNAs) for antisense and single nucleotide polymorphism detection

    NARCIS (Netherlands)

    Gahtory, Digvijay; Murtola, Merita; Smulders, Maarten M.J.; Wennekes, Tom; Zuilhof, Han; Strömberg, Roger; Albada, Bauke

    2017-01-01

    In this report, we show how a convenient on-resin copper-click functionalization of azido-functionalized peptide nucleic acids (PNAs) allows various PNA-based detection strategies. Firstly, a thiazole orange (TO) clicked PNA probe facilitates a binary readout when combined with F/Q labeled DNA,

  3. Modulation of i-motif thermodynamic stability by the introduction of UNA (unlocked nucleic acid) monomers

    DEFF Research Database (Denmark)

    Pasternak, Anna; Wengel, Jesper

    2011-01-01

    The influence of acyclic RNA derivatives, UNA (unlocked nucleic acid) monomers, on i-DNA thermodynamic stability has been investigated. The 22 nt human telomeric fragment was chosen as the model sequence for stability studies. UNA monomers modulate i-motif stability in a position-depending manner...

  4. The Chemistry of Polymers, Proteins, and Nucleic Acids: A Short Course on Macromolecules for Secondary Schools.

    Science.gov (United States)

    Lulav, Ilan; Samuel, David

    1985-01-01

    Describes a unit on macromolecules that has been used in the 12th grade of many Israeli secondary schools. Topic areas in the unit include synthetic polymers, biological macromolecules, and nucleic acids. A unit outline is provided in an appendix. (JN)

  5. Modification of nucleic acids by azobenzene derivatives and their applications in biotechnology and nanotechnology.

    Science.gov (United States)

    Li, Jing; Wang, Xingyu; Liang, Xingguo

    2014-12-01

    Azobenzene has been widely used as a photoregulator due to its reversible photoisomerization, large structural change between E and Z isomers, high photoisomerization yield, and high chemical stability. On the other hand, some azobenzene derivatives can be used as universal quenchers for many fluorophores. Nucleic acid is a good candidate to be modified because it is not only the template of gene expression but also widely used for building well-organized nanostructures and nanodevices. Because the size and polarity distribution of the azobenzene molecule is similar to a nucleobase pair, the introduction of azobenzene into nucleic acids has been shown to be an ingenious molecular design for constructing light-switching biosystems or light-driven nanomachines. Here we review recent advances in azobenzene-modified nucleic acids and their applications for artificial regulation of gene expression and enzymatic reactions, construction of photoresponsive nanostructures and nanodevices, molecular beacons, as well as obtaining structural information using the introduced azobenzene as an internal probe. In particular, nucleic acids bearing multiple azobenzenes can be used as a novel artificial nanomaterial with merits of high sequence specificity, regular duplex structure, and high photoregulation efficiency. The combination of functional groups with biomolecules may further advance the development of chemical biotechnology and biomolecular engineering. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Synthetic Nucleic Acid Analogues in Gene Therapy: An Update for Peptide–Oligonucleotide Conjugates

    DEFF Research Database (Denmark)

    Taskova, Maria; Mantsiou, Anna; Astakhova, Kira

    2017-01-01

    The main objective of this work is to provide an update on synthetic nucleic acid analogues and nanoassemblies as tools in gene therapy. In particular, the synthesis and properties of peptide–oligonucleotide conjugates (POCs), which have high potential in research and as therapeutics, are described...

  7. Recent Advances in Nucleic Acid Targeting Probes and Supramolecular Constructs Based on Pyrene-Modified Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Olga A. Krasheninina

    2017-11-01

    Full Text Available In this review, we summarize the recent advances in the use of pyrene-modified oligonucleotides as a platform for functional nucleic acid-based constructs. Pyrene is of special interest for the development of nucleic acid-based tools due to its unique fluorescent properties (sensitivity of fluorescence to the microenvironment, ability to form excimers and exciplexes, long fluorescence lifetime, high quantum yield, ability to intercalate into the nucleic acid duplex, to act as a π-π-stacking (including anchoring moiety, and others. These properties of pyrene have been used to construct novel sensitive fluorescent probes for the sequence-specific detection of nucleic acids and the discrimination of single nucleotide polymorphisms (SNPs, aptamer-based biosensors, agents for binding of double-stranded DNAs, and building blocks for supramolecular complexes. Special attention is paid to the influence of the design of pyrene-modified oligonucleotides on their properties, i.e., the structure-function relationships. The perspectives for the applications of pyrene-modified oligonucleotides in biomolecular studies, diagnostics, and nanotechnology are discussed.

  8. Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

    DEFF Research Database (Denmark)

    Birkedal, Henrik; Nielsen, Peter E

    2011-01-01

    Gene correction activation effects of a small series of triplex forming peptide nucleic acid (PNA) covalently conjugated to the DNA interacting ligands psoralen, chlorambucil and camptothecin targeted proximal to a stop codon mutation in an EGFP reporter gene were studied. A 15-mer homopyrimidine...

  9. Extracellular vesicles for nucleic acid delivery : progress and prospects for safe RNA-based gene therapy

    NARCIS (Netherlands)

    Jiang, L; Vader, P; Schiffelers, R M

    2017-01-01

    Nucleic acid-based drugs offer a potentially effective tool for treatment of a variety of diseases, including cancer, cardiovascular diseases, neurological disorders and infectious diseases. However, clinical applications are hindered by instability of RNA molecules in the circulation and lack of

  10. 77 FR 16126 - Microbiology Devices; Reclassification of Nucleic Acid-Based Systems for Mycobacterium tuberculosis

    Science.gov (United States)

    2012-03-19

    ... Food and Drug Administration 21 CFR Part 866 Microbiology Devices; Reclassification of Nucleic Acid... the Microbiology Devices Panel of the Medical Devices Advisory Committee (Microbiology Devices Panel.... VI. Risks to Health After considering the information discussed by the Microbiology Devices Panel...

  11. The Prebiotic Synthesis of Ethylenediamine Monoacetic Acid, The Repeating Unit of Peptide Nucleic Acids

    Science.gov (United States)

    Nelson, Kevin E.; Miller, Stanley L.

    1992-01-01

    The polymerization of ribonucleic acids or their precursors constitutes an important event in prebiotic chemistry. The various problems using ribonucleotides to make RNA suggest that there may have been a precursor. An attractive possibility are the peptide nucleic acids (PNA). PNAs are nucleotide analogs that make use of a polymer of ethylenediamine monoacetic acid (EDMA or 2-amninoethyl glycine) with the bases attached by an acetic acid. EDMA is an especially attractive alternative to the ribose phosphate or deoxyribose phosphate backbone because it contains no chiral centers and is potentially prebiotic, but there is no reported prebiotic synthesis. We have synthesized both EDMA and ethylenediamine diacetic acid (EDDA) from the prebiotic compounds ethylenediamine, formaldehyde, and hydrogen cyanide. The yields of EDMA range from 11 to 79% along with some sEDDA and uEDDA. These reactions work with concentrations of 10(exp -1)M and as low as 10(exp -4)M, and the reaction is likely to be effective at even lower concentrations. Ethylenediamine is a likely prebiotic compound, but it has not yet been demonstrated, although compounds such as ethanolamine and cysteamine have been proven to be prebiotic. Under neutral pH and heating at l00 C, EDMA is converted to the lactam, monoketopiperazine (MKP). The cyclization occurs and has an approximate ratio of MKP/EDMA = 3 at equilibrium. We have measured the solubilities of EDMA center dot H20 as 6.4 m, EDMA center dot HCl center dot H20 as 13.7 m, and EDMA center dot 2HCl center dot H20 as 3.4 m. These syntheses together with the high solubility of EDMA suggest that EDMA would concentrate in drying lagoons and might efficiently form polymers. Given the instability of ribose and the poor polymerizability of nucleotides, the prebiotic presence of EDMA and the possibility of its polymerization raises the possibility that PNAs are the progenitors of present day nucleic acids. A pre-RNA world may have existed in which PNAs or

  12. Detection of nucleic acid-protein interactions in plant leaves using fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Camborde, Laurent; Jauneau, Alain; Brière, Christian; Deslandes, Laurent; Dumas, Bernard; Gaulin, Elodie

    2017-09-01

    DNA-binding proteins (DNA-BPs) and RNA-binding proteins (RNA-BPs) have critical roles in living cells in all kingdoms of life. Various experimental approaches exist for the study of nucleic acid-protein interactions in vitro and in vivo, but the detection of such interactions at the subcellular level remains challenging. Here we describe how to detect nucleic acid-protein interactions in plant leaves by using a fluorescence resonance energy transfer (FRET) approach coupled to fluorescence lifetime imaging microscopy (FLIM). Proteins of interest (POI) are tagged with a GFP and transiently expressed in plant cells to serve as donor fluorophore. After sample fixation and cell wall permeabilization, leaves are treated with Sytox Orange, a nucleic acid dye that can function as a FRET acceptor. Upon close association of the GFP-tagged POI with Sytox-Orange-stained nucleic acids, a substantial decrease of the GFP lifetime due to FRET between the donor and the acceptor can be monitored. Treatment with RNase before FRET-FLIM measurements allows determination of whether the POI associates with DNA and/or RNA. A step-by-step protocol is provided for sample preparation, data acquisition and analysis. We describe how to calibrate the equipment and include a tutorial explaining the use of the FLIM software. To illustrate our approach, we provide experimental procedures to detect the interaction between plant DNA and two proteins (the AeCRN13 effector from the oomycete Aphanomyces euteiches and the AtWRKY22 defensive transcription factor from Arabidopsis). This protocol allows the detection of protein-nucleic acid interactions in plant cells and can be completed in <2 d.

  13. Geometric Patterns for Neighboring Bases Near the Stacked State in Nucleic Acid Strands.

    Science.gov (United States)

    Sedova, Ada; Banavali, Nilesh K

    2017-03-14

    Structural variation in base stacking has been analyzed frequently in isolated double helical contexts for nucleic acids, but not as often in nonhelical geometries or in complex biomolecular environments. In this study, conformations of two neighboring bases near their stacked state in any environment are comprehensively characterized for single-strand dinucleotide (SSD) nucleic acid crystal structure conformations. An ensemble clustering method is used to identify a reduced set of representative stacking geometries based on pairwise distances between select atoms in consecutive bases, with multiple separable conformational clusters obtained for categories divided by nucleic acid type (DNA/RNA), SSD sequence, stacking face orientation, and the presence or absence of a protein environment. For both DNA and RNA, SSD conformations are observed that are either close to the A-form, or close to the B-form, or intermediate between the two forms, or further away from either form, illustrating the local structural heterogeneity near the stacked state. Among this large variety of distinct conformations, several common stacking patterns are observed between DNA and RNA, and between nucleic acids in isolation or in complex with proteins, suggesting that these might be stable stacking orientations. Noncanonical face/face orientations of the two bases are also observed for neighboring bases in the same strand, but their frequency is much lower, with multiple SSD sequences across categories showing no occurrences of such unusual stacked conformations. The resulting reduced set of stacking geometries is directly useful for stacking-energy comparisons between empirical force fields, prediction of plausible localized variations in single-strand structures near their canonical states, and identification of analogous stacking patterns in newly solved nucleic acid containing structures.

  14. Lateral flow devices for nucleic acid analysis exploiting quantum dots as reporters

    Energy Technology Data Exchange (ETDEWEB)

    Sapountzi, Eleni A.; Tragoulias, Sotirios S.; Kalogianni, Despina P. [Department of Chemistry, University of Patras, GR-26504 Patras (Greece); Ioannou, Penelope C. [Department of Chemistry, University of Athens, GR-15771 Athens (Greece); Christopoulos, Theodore K., E-mail: tchrist@upatras.gr [Department of Chemistry, University of Patras, GR-26504 Patras (Greece); Institute of Chemical Engineering and High Temperature Processes, Foundation of Research and Technology Hellas, GR-26504 Patras (Greece)

    2015-03-15

    Highlights: • Dipstick tests for DNA hybridization assays and genotyping of single-nucleotide polymorphisms. • Use of quantum dots as reporters. • Visual detection without the need for expensive instrumentation. • Simplicity and low-cost of the assays. - Abstract: There is a growing interest in the development of biosensors in the form of simple lateral flow devices that enable visual detection of nucleic acid sequences while eliminating several steps required for pipetting, incubation and washing out the excess of reactants. In this work, we present the first dipstick-type nucleic acid biosensors based on quantum dots (QDs) as reporters. The biosensors enable sequence confirmation of the target DNA by hybridization and simple visual detection of the emitted fluorescence under a UV lamp. The ‘diagnostic’ membrane of the biosensor contains a test zone (TZ) and a control zone (CZ). The CZ always fluoresces in order to confirm the proper function of the biosensor. Fluorescence is emitted from the TZ, only when the specific nucleic acid sequence is present. We have developed two general types of QD-based nucleic acid biosensors, namely, Type I and Type II, in which the TZ consists of either immobilized streptavidin (Type I) or immobilized oligodeoxynucleotides (Type II). The control zone consists of immobilized biotinylated albumin. No purification steps are required prior to the application of the DNA sample on the strip. The QD-based nucleic acid biosensors performed accurately and reproducibly when applied to (a) the visual detection of PCR amplification products and (b) visual genotyping of single nucleotide polymorphisms (SNPs) in human genomic DNA from clinical samples. As low as 1.5 fmol of double-stranded DNA were clearly detected by naked eye and the dynamic range extended to 200 fmol. The %CV were estimated to be 4.3–8.2.

  15. The 2016 database issue of Nucleic Acids Research and an updated molecular biology database collection.

    Science.gov (United States)

    Rigden, Daniel J; Fernández-Suárez, Xosé M; Galperin, Michael Y

    2016-01-04

    The 2016 Database Issue of Nucleic Acids Research starts with overviews of the resources provided by three major bioinformatics centers, the U.S. National Center for Biotechnology Information (NCBI), the European Bioinformatics Institute (EMBL-EBI) and Swiss Institute for Bioinformatics (SIB). Also included are descriptions of 62 new databases and updates on 95 databases that have been previously featured in NAR plus 17 previously described elsewhere. A number of papers in this issue deal with resources on nucleic acids, including various kinds of non-coding RNAs and their interactions, molecular dynamics simulations of nucleic acid structure, and two databases of super-enhancers. The protein database section features important updates on the EBI's Pfam, PDBe and PRIDE databases, as well as a variety of resources on pathways, metabolomics and metabolic modeling. This issue also includes updates on popular metagenomics resources, such as MG-RAST, EBI Metagenomics, and probeBASE, as well as a newly compiled Human Pan-Microbe Communities database. A significant fraction of the new and updated databases are dedicated to the genetic basis of disease, primarily cancer, and various aspects of drug research, including resources for patented drugs, their side effects, withdrawn drugs, and potential drug targets. A further six papers present updated databases of various antimicrobial and anticancer peptides. The entire Database Issue is freely available online on the Nucleic Acids Research website (http://nar.oxfordjournals.org/). The NAR online Molecular Biology Database Collection, http://www.oxfordjournals.org/nar/database/c/, has been updated with the addition of 88 new resources and removal of 23 obsolete websites, which brought the current listing to 1685 databases. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  16. The relationship between odd- and branched-chain fatty acids and microbial nucleic acid bases in rumen.

    Science.gov (United States)

    Liu, Keyuan; Hao, Xiaoyan; Li, Yang; Luo, Guobin; Zhang, Yonggen; Xin, Hangshu

    2017-11-01

    This study aims to identify the relationship between odd- and branched-chain fatty acids (OBCFAs) and microbial nucleic acid bases in the rumen, and to establish a model to accurately predict microbial protein flow by using OBCFA. To develop the regression equations, data on the rumen contents of individual cows were obtained from 2 feeding experiments. In the first experiment, 3 rumen-fistulated dry dairy cows arranged in a 3×3 Latin square were fed diets of differing forage to concentration ratios (F:C). The second experiment consisted of 9 lactating Holstein dairy cows of similar body weights at the same stage of pregnancy. For each lactation stage, 3 cows with similar milk production were selected. The rumen contents were sampled at 4 time points of every two hours after morning feeding 6 h, and then to analyse the concentrations of OBCFA and microbial nucleic acid bases in the rumen samples. The ruminal bacteria nucleic acid bases were significantly influenced by feeding diets of differing forge to concentration ratios and lactation stages of dairy cows (pacids and C15:0 isomers, strongly correlated with the microbial nucleic acid bases in the rumen (pacid bases established by ruminal OBCFAs contents showed a good predictive capacity, as indicated by reasonably low standard errors and high R-squared values. This finding suggests that the rumen OBCFA composition could be used as an internal marker of rumen microbial matter.

  17. The relationship between odd- and branched-chain fatty acids and microbial nucleic acid bases in rumen

    Directory of Open Access Journals (Sweden)

    Keyuan Liu

    2017-11-01

    Full Text Available Objective This study aims to identify the relationship between odd- and branched-chain fatty acids (OBCFAs and microbial nucleic acid bases in the rumen, and to establish a model to accurately predict microbial protein flow by using OBCFA. Methods To develop the regression equations, data on the rumen contents of individual cows were obtained from 2 feeding experiments. In the first experiment, 3 rumen-fistulated dry dairy cows arranged in a 3×3 Latin square were fed diets of differing forage to concentration ratios (F:C. The second experiment consisted of 9 lactating Holstein dairy cows of similar body weights at the same stage of pregnancy. For each lactation stage, 3 cows with similar milk production were selected. The rumen contents were sampled at 4 time points of every two hours after morning feeding 6 h, and then to analyse the concentrations of OBCFA and microbial nucleic acid bases in the rumen samples. Results The ruminal bacteria nucleic acid bases were significantly influenced by feeding diets of differing forge to concentration ratios and lactation stages of dairy cows (p<0.05. The concentrations of OBCFAs, especially odd-chain fatty acids and C15:0 isomers, strongly correlated with the microbial nucleic acid bases in the rumen (p<0.05. The equations of ruminal microbial nucleic acid bases established by ruminal OBCFAs contents showed a good predictive capacity, as indicated by reasonably low standard errors and high R-squared values. Conclusion This finding suggests that the rumen OBCFA composition could be used as an internal marker of rumen microbial matter.

  18. Sample to answer: a fully integrated nucleic acid identification system for bacteria monitoring

    Science.gov (United States)

    Kim, Jungkyu; Elsnab, John; Johnson, Michael; Gale, Bruce K.

    2010-02-01

    A fully integrated microfluidic system was developed and incorporates an EC-MWCNT (electrochemical multiwalled carbon nanotube) sensor for the detection of bacteria. Sample metering, reagent metering and delivery was implemented with microvalves and pumps embedded inside the microfluidic system. The nucleic acid extraction was performed using microchannels controlled using automated platforms and a disposable microfluidic silica cartridge. The target samples were flowed and hybridized with probe ssDNA (single strand DNA) across the MWCNT-EC sensor (built on a silicon chip), which was embedded in a microfluidic cell. The 9-pad sensor was scanned before and after hybridization to measure the quantity of RNA (Ribonucleic acid) bound to the array surface. A rapid and accurate sample-in answer-out nucleic acid system was realized with automated volume metering, microfluidic sample preparation, and integrated nano-biosensors.

  19. Adsorption and isolation of nucleic acids on cellulose magnetic beads using a three-dimensional printed microfluidic chip.

    Science.gov (United States)

    Zhang, Lei; Deraney, Rachel N; Tripathi, Anubhav

    2015-11-01

    While advances in genomics have enabled sensitive and highly parallel detection of nucleic acid targets, the isolation and extraction of the nucleic acids remain a critical bottleneck in the workflow. We present here a simple 3D printed microfluidic chip that allows for the vortex and centrifugation free extraction of nucleic acids. This novel microfluidic chip utilizes the presence of a water and oil interface to filter out the lysate contaminants. The pure nucleic acids, while bound on cellulose particles, are magnetically moved across the oil layer. We demonstrated efficient and rapid extraction of spiked Human Papillomavirus (HPV) 18 plasmids in specimen transport medium, in under 15 min. An overall extraction efficiency of 61% is observed across a range of HPV plasmid concentrations (5 × 10(1) to 5 × 10(6) copies/100 μl). The magnetic, interfacial, and viscous drag forces inside the microgeometries of the chip are modeled. We have also developed a kinetics model for the adsorption of nucleic acids on cellulose functionalized superparamagnetic beads. We also clarify here the role of carrier nucleic acids in the adsorption and isolation of nucleic acids. Based on the various mechanistic insights detailed here, customized microfluidic devices can be designed to meet the range of current and emerging point of care diagnostics needs.

  20. Nucleic Acid Sensing Machinery: Targeting Innate Immune System for Cancer Therapy.

    Science.gov (United States)

    Iurescia, Sandra; Fioretti, Daniela; Rinaldi, Monica

    2017-10-30

    • Background Nucleic acid sensing is an essential strategy employed by the innate immune system to detect both pathogen-derived nucleic acids and self-DNA released by host apoptotic or necrotic cells. The presence of nucleic acids that gain access to the cytoplasm is perceived by mammalian cells as "stranger" or "danger" signals that triggers a myriad of immunological responses. Recent publications have highlighted the importance of nucleic acid sensing machinery as mediator of innate and adaptive immunity, and cGAS, STING and RIG-I agonists have been validated as immuno-oncology agents in cancer therapy. • Objective The crucial role of cGAS and STING in eliciting innate and adaptive immune responses provides a scientific rationale for using cGAMP and STING agonists both in human preventive vaccine and immunotherapy settings. Thus, search for natural and synthetic STING agonists and development of cyclic dinucleotides (CDNs)-based adjuvants were strongly intensified. Furthermore, with their ability to induce tumour cell death and lymphocyte cross priming, RIG-I ligands are among the most promising molecules for the development of new immunostimulatory adjuvants in cancer vaccines. • Results This work focuses on relevant recent patents (2010-2017) that entail the use of nucleic acids sensing machinery to elicit innate and adaptive immune responses, highlighting a new approach in immune-mediated cancer therapy. Several patents describe compositions and methods that may be used as immuno-oncology agents for treatment of cancer patients. cGAS and/or STING pathways modulating compounds alone or in combination with pharmaceutical compositions are discussed. New approaches to improve DNA-vaccine induced adaptive immunity for cancer therapy through increasing level of plasmid-mediated activation of innate immune signalling pathways are also discussed. In addition, a targeted selection of very recent clinical studies describing the employment of innate immunity

  1. Macro-/Nano- Materials Based Ultrasensitive Lateral Flow Nucleic Acid Biosensors

    Science.gov (United States)

    Takalkar, Sunitha

    Ultrasensitive detection of nucleic acids plays a very important role in the field of molecular diagnosis for the detection of various diseases. Lateral flow biosensors (LFB) are convenient, easy-to-use, patient friendly forms of detection methods offering rapid and convenient clinical testing in close proximity to the patients thus drawing a lot of attention in different areas of research over the years. In comparison with the traditional immunoassays, the nucleic acid based lateral flow biosensors (NABLFB) has several advantages in terms of stability and interference capabilities. NABLFB utilizes nucleic acid probes as the bio-recognition element. The target analyte typically is the oligonucleotide like the DNA, mRNA, miRNA which are among the nucleic acid secretions by the tumor cells when it comes to detection of cancer. Traditionally gold nanoparticles (GNPs) have been used as labels for conjugating with the detection probes for the qualitative and semi quantitative analysis, the application of GNP-based LFB is limited by its low sensitivity. This dissertation describes the use of different nanomaterials and advanced detection technologies to enhance the sensitivities of the LFB based methods. Silica Nanorods decorated with GNP were synthesized and employed as labels for ultrasensitive detection of miRNA on the LFB. Owing to the biocompatibility and convenience in surface modification of SiNRs, they acted as good carriers to load numerous GNPs. The sensitivity of the GNP-SiNR-based LFSB was enhanced six times compared to the previous GNP-based LFSB. A fluorescent carbon nanoparticle (FCN) was first used as a tag to develop a lateral flow nucleic acid biosensor for ultrasensitive and quantitative detection of nucleic acid samples. Under optimal conditions, the FCN-based LFNAB was capable of detecting minimum 0.4 fM target DNA without complex operations and additional signal amplification. The carbon nanotube was used as a label and carrier of numerous enzyme

  2. Molecular Recognition and Structural Influences on Function in Bio-nanosystems of Nucleic Acids and Proteins

    Science.gov (United States)

    Sethaphong, Latsavongsakda

    This work examines smart material properties of rational self-assembly and molecular recognition found in nano-biosystems. Exploiting the sequence and structural information encoded within nucleic acids and proteins will permit programmed synthesis of nanomaterials and help create molecular machines that may carry out new roles involving chemical catalysis and bioenergy. Responsive to different ionic environments thru self-reorgnization, nucleic acids (NA) are nature's signature smart material; organisms such as viruses and bacteria use features of NAs to react to their environment and orchestrate their lifecycle. Furthermore, nucleic acid systems (both RNA and DNA) are currently exploited as scaffolds; recent applications have been showcased to build bioelectronics and biotemplated nanostructures via directed assembly of multidimensional nanoelectronic devices 1. Since the most stable and rudimentary structure of nucleic acids is the helical duplex, these were modeled in order to examine the influence of the microenvironment, sequence, and cation-dependent perturbations of their canonical forms. Due to their negatively charged phosphate backbone, NA's rely on counterions to overcome the inherent repulsive forces that arise from the assembly of two complementary strands. As a realistic model system, we chose the HIV-TAR helix (PDB ID: 397D) to study specific sequence motifs on cation sequestration. At physiologically relevant concentrations of sodium and potassium ions, we observed sequence based effects where purine stretches were adept in retaining high residency cations. The transitional space between adenine and guanosine nucleotides (ApG step) in a sequence proved the most favorable. This work was the first to directly show these subtle interactions of sequence based cationic sequestration and may be useful for controlling metallization of nucleic acids in conductive nanowires. Extending the study further, we explored the degree to which the structure of NA

  3. Coronavirus phylogeny based on triplets of nucleic acids bases

    Science.gov (United States)

    Liao, Bo; Liu, Yanshu; Li, Renfa; Zhu, Wen

    2006-04-01

    We considered the fully overlapping triplets of nucleotide bases and proposed a 2D graphical representation of protein sequences consisting of 20 amino acids and a stop code. Based on this 2D graphical representation, we outlined a new approach to analyze the phylogenetic relationships of coronaviruses by constructing a covariance matrix. The evolutionary distances are obtained through measuring the differences among the two-dimensional curves.

  4. Method and apparatus for purifying nucleic acids and performing polymerase chain reaction assays using an immiscible fluid

    Science.gov (United States)

    Koh, Chung-Yan; Light, Yooli Kim; Piccini, Matthew Ernest; Singh, Anup K.

    2017-10-31

    Embodiments of the present invention are directed toward devices, systems, and methods for purifying nucleic acids to conduct polymerase chain reaction (PCR) assays. In one example, a method includes generating complexes of silica beads and nucleic acids in a lysis buffer, transporting the complexes through an immiscible fluid to remove interfering compounds from the complexes, further transporting the complexes into a density medium containing components required for PCR where the nucleic acids disassociate from the silica beads, and thermocycling the contents of the density medium to achieve PCR. Signal may be detected from labeling agents in the components required for PCR.

  5. Nonenzymatic synthesis of RNA and DNA oligomers on hexitol nucleic acid templates: the importance of the A structure

    Science.gov (United States)

    Kozlov, I. A.; Politis, P. K.; Van Aerschot, A.; Busson, R.; Herdewijn, P.; Orgel, L. E.; Bada, J. L. (Principal Investigator); Dolan, M. (Principal Investigator)

    1999-01-01

    Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.

  6. Evaluation of automated nucleic acid extraction methods for virus detection in a multicenter comparative trial

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Hakhverdyan, M.

    2009-01-01

    laboratory extracted nucleic acid from this panel using available robotic equipment (12 separate instruments, comprising 8 different models), after which the processed samples were frozen and sent to a single laboratory for subsequent testing by real-time RT-PCR. In general, there was good concordance...... between the results obtained for the different automated extraction platforms. In particular, the limit of detection was identical for 9/12 and 8/12 best performing robots (using dilutions of BVDV infected-serum and cell culture material, respectively), which was similar to a manual extraction method used......Five European veterinary laboratories participated in an exercise to compare the performance of nucleic acid extraction robots. Identical sets of coded samples were prepared using serial dilutions of bovine viral diarrhoea virus (BVDV) from serum and cell culture propagated material. Each...

  7. Rapid Bedside Inactivation of Ebola Virus for Safe Nucleic Acid Tests

    DEFF Research Database (Denmark)

    Rosenstierne, Maiken Worsøe; Karlberg, Helen; Bragstad, Karoline

    2016-01-01

    for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding Magna Pure lysis/binding buffer directly into vacuum blood collection EDTA tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum......Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport, and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available Magna Pure lysis/binding buffer used...... tubes are stable for more than 4 months, and Ebola virus RNA is preserved in the Magna Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from Magna Pure lysis/binding buffer-inactivated samples using...

  8. Preparation of surfactant-stabilized gold nanoparticle-peptide nucleic acid conjugates

    Energy Technology Data Exchange (ETDEWEB)

    Duy, Janice, E-mail: janice.duy@umit.maine.ed [University of Maine, Graduate School of Biomedical Sciences (United States); Connell, Laurie B. [University of Maine, School of Marine Sciences (United States); Eck, Wolfgang [University of Heidelberg, Applied Physical Chemistry (Germany); Collins, Scott D. [University of Maine, Department of Chemistry (United States); Smith, Rosemary L. [University of Maine, Electrical and Computer Engineering Department (United States)

    2010-09-15

    A simple, two-step method of producing stable and functional peptide nucleic acid (PNA)-conjugated gold nanoparticles using a surfactant stabilization step is presented. PNA are DNA analogs with superior chemical stability and target discrimination, but their use in metallic nanoparticle systems has been limited by the difficulty of producing stable colloids of nanoparticle-PNA conjugates. In this work, the nonionic surfactant Tween 20 (polyoxyethylene (20) sorbitan monolaurate) was used to sterically shield gold surfaces prior to the addition of thiolated PNA, producing conjugates which remain dispersed in solution and retain the ability to hybridize to complementary nucleic acid sequences. The conjugates were characterized using transmission electron microscopy, dynamic light scattering, and UV-visible absorbance spectroscopy. PNA attachment to gold nanoparticles was confirmed with an enzyme-linked immunoassay, while the ability of nanoparticle-bound PNA to hybridize to its complement was demonstrated using labeled DNA.

  9. [Advancement in the research of early detection of bacterial nucleic acid in molecular diagnosis of sepsis].

    Science.gov (United States)

    Liu, Xiao; Ren, Hui; Peng, Dai-zhi

    2013-04-01

    Early diagnosis of sepsis helps make effective clinical decisions and improve the survival rate of patients with severe infection. However, the timely and accurate diagnosis of sepsis is still a great challenge in clinic. In order to settle the very problem, the scientists in the world have made a lot of exploration and research in the field of rapid molecular identification of pathogens. Nowadays, the nucleic acid detection of sepsis is mainly composed of 3 types of methodological strategies, either based on positive blood culture, single colonies, or directly on blood specimens. This paper presents a comprehensive overview of advances in the research of early detection of bacterial nucleic acid as molecular diagnosis of sepsis.

  10. Amplicon Competition Enables End-Point Quantitation of Nucleic Acids Following Isothermal Amplification.

    Science.gov (United States)

    Jiang, Yu Sherry; Stacy, Apollo; Whiteley, Marvin; Ellington, Andrew D; Bhadra, Sanchita

    2017-09-05

    It is inherently difficult to quantitate nucleic acid analytes with most isothermal amplification assays. We developed loop-mediated isothermal amplification (LAMP) reactions in which competition between defined numbers of "false" and "true" amplicons leads to order of magnitude quantitation by a single endpoint determination. These thresholded LAMP reactions were successfully used to directly and quantitatively estimate the numbers of nucleic acids in complex biospecimens, including directly from cells and in sewage, with the values obtained closely correlating with qPCR quantitations. Thresholded LAMP reactions are amenable to endpoint readout by cell phone, unlike other methods that require continuous monitoring, and should therefore prove extremely useful in developing one-pot reactions for point-of-care diagnostics without needing sophisticated material or informatics infrastructure. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Single-molecule pull-down for investigating protein-nucleic acid interactions.

    Science.gov (United States)

    Fareh, Mohamed; Loeff, Luuk; Szczepaniak, Malwina; Haagsma, Anna C; Yeom, Kyu-Hyeon; Joo, Chirlmin

    2016-08-01

    The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids. Here, we combined single-molecule fluorescence with various protein complex pull-down techniques to determine the function and stoichiometry of ribonucleoprotein complexes. Through the use of three examples of protein complexes from eukaryotic cells (Drosha, Dicer, and TUT4 protein complexes), we provide step-by-step guidance for using novel single-molecule techniques. Our single-molecule methods provide sub-second and nanometer resolution and can be applied to other nucleoprotein complexes that are essential for cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. SDS-PAGE procedure: Application for characterization of new entirely uncharged nucleic acids analogs.

    Science.gov (United States)

    Pavlova, Anna S; Dyudeeva, Evgeniya S; Kupryushkin, Maxim S; Amirkhanov, Nariman V; Pyshnyi, Dmitrii V; Pyshnaya, Inna A

    2018-02-01

    SDS-PAGE is considered to be a universal method for size-based separation and analysis of proteins. In this study, we applied the principle of SDS-PAGE to the analysis of new entirely uncharged nucleic acid (NA) analogues, - phosphoryl guanidine oligonucleotides (PGOs). The procedure was also shown to be suitable for morpholino oligonucleotides (PMOs) and peptide nucleic acids (PNAs). It was demonstrated that SDS can establish hydrophobic interactions with these types of synthetic NAs, giving them a net negative charge and thus making these molecules mobile in polyacrylamide slab gels under the influence of an electric field. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Simultaneous detection of nucleic acid and protein using gold nanoparticles and lateral flow device.

    Science.gov (United States)

    Mao, Xun; Gurung, Anant; Xu, Hui; Baloda, Meenu; He, Yuqing; Liu, Guodong

    2014-01-01

    In this work, we present a simple and fast approach for simultaneous detection of nucleic acid and protein using gold nanoparticles (GNPs) and a lateral flow device (LFD). Sandwich-type immunoreactions and DNA hybridizations were performed simultaneously on the LFD by using DNA- and antibody-functionalized GNPs. The captured GNPs, due to the DNA hybridization and immunoreaction events on the LFD, produced characteristic red bands that could be used for the qualitative detections of DNA and/or protein. The proof of principle was demonstrated by using 60-mer DNA and rabbit IgG (R-IgG) model targets. The LFD was capable of detecting a minimum of 0.5 nM target DNA and 2 ng mL(-1) IgG simultaneously in 15 min. The proposed LFD shows great promise for in-field and point-of-care testing of disease-related circulating nucleic acid and protein biomarkers in biological fluids.

  14. Synthetic oligonucleotide antigens modified with locked nucleic acids detect disease specific antibodies

    Science.gov (United States)

    Samuelsen, Simone V.; Solov'Yov, Ilia A.; Balboni, Imelda M.; Mellins, Elizabeth; Nielsen, Christoffer Tandrup; Heegaard, Niels H. H.; Astakhova, Kira

    2016-10-01

    New techniques to detect and quantify antibodies to nucleic acids would provide a significant advance over current methods, which often lack specificity. We investigate the potential of novel antigens containing locked nucleic acids (LNAs) as targets for antibodies. Particularly, employing molecular dynamics we predict optimal nucleotide composition for targeting DNA-binding antibodies. As a proof of concept, we address a problem of detecting anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We test the best oligonucleotide binders in surface plasmon resonance studies to analyze binding and kinetic aspects of interactions between antigens and target DNA. These DNA and LNA/DNA sequences showed improved binding in enzyme-linked immunosorbent assay using human samples of pediatric lupus patients. Our results suggest that the novel method is a promising tool to create antigens for research and point-of-care monitoring of anti-DNA antibodies.

  15. Magnetic bead-based nucleic acid purification kit: Clinical application and performance evaluation in stool specimens.

    Science.gov (United States)

    Yoon, Jihoon G; Kang, Jin Seok; Hwang, Seung Yong; Song, Jaewoo; Jeong, Seok Hoon

    2016-05-01

    Two different methods - the semi-automated magnetic bead-based kit (SK, Stool DNA/RNA Purification kit®) and the manual membrane column-based kit (QS, QIAamp® DNA Stool Mini kit) - for purifying nucleic acids from clinical stool samples were compared and evaluated. The SK kit was more user-friendly than QS due to the reduced manual processing, partial automation, and short turnaround time with half cost. Furthermore, SK produced high yields in both DNA and RNA extractions but poor purity in RNA extraction. In the assessment of rotavirus and Clostridium difficile infection, both kits had equivalent or more sensitive performance compared with the standard method. Although SK showed some interference and inhibition in nucleic acid extraction, the performance, including the repeatability, linearity, analytical sensitivity, and matrix effect, was sufficient for routine clinical use. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Peripheral Blood Epstein-Barr Viral Nucleic Acid Surveillance as a Marker for Posttransplant Cancer Risk.

    Science.gov (United States)

    Dharnidharka, V R

    2017-03-01

    Several viruses, such as Epstein-Barr virus, are now known to be associated with several human cancers, but not all patients with these viral infections develop cancer. In transplantation, such viruses often have a prolonged time gap from infection to cancer development, and many are preceded by a period of circulating and detectable nucleic acids in the peripheral blood compartment. The interpretation of a viral load as a measure of posttransplant risk of developing cancer depends on the virus, the cancer and associated pathogenic factors. This review describes the current state of knowledge regarding the utility and limitations of peripheral blood nucleic acid testing for Epstein-Barr virus in surveillance and risk prediction for posttransplant lymphoproliferative disorders. © Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

  17. Preliminary Classification of Viruses Based on Quantitative Comparisons of Viral Nucleic Acids

    Science.gov (United States)

    Bellett, A. J. D.

    1967-01-01

    It is proposed that classifications used in science are of two main types; those which are designed to solve practical problems and which are based on conventions, and those which are designed to solve theoretical problems, based on theories, and in which the classes are tested by experiment. An attempt has been made to construct a preliminary classification of viruses which is of the second type. It is based on the theories of molecular biology, with the use of computer-based comparisons of the molecular weights and base ratios of viral nucleic acids to assign the viruses to clusters which show a high degree of correlation with groupings based on nucleic acid hybridization, serological cross-reactions, and phenotypic properties. Images PMID:5623961

  18. Synthesis and degradation of nucleic acid components by formamide and iron sulfur minerals.

    Science.gov (United States)

    Saladino, Raffaele; Neri, Veronica; Crestini, Claudia; Costanzo, Giovanna; Graciotti, Michele; Di Mauro, Ernesto

    2008-11-19

    We describe the one-pot synthesis of a large panel of nucleic bases and related compounds from formamide in the presence of iron sulfur and iron-copper sulfur minerals as catalysts. The major products observed are purine, 1H-pyrimidinone, isocytosine, adenine, 2-aminopurine, carbodiimide, urea, and oxalic acid. Isocytosine and 2-aminopurine may recognize natural nucleobases by Watson-Crick and reverse Watson-Crick interactions, thus suggesting novel scenarios for the origin of primordial nucleic acids. Since the major problem in the origin of informational polymers is the instability of their precursors, we also investigate the effects of iron sulfur and iron-copper sulfur minerals on the stability of ribooligonucleotides in formamide and in water. All of the iron sulfur and iron-copper sulfur minerals stimulated degradation of RNA. The relevance of these findings with respect to the origin of informational polymers is discussed.

  19. The Use of Atomic Force Microscopy for 3D Analysis of Nucleic Acid Hybridization on Microarrays.

    Science.gov (United States)

    Dubrovin, E V; Presnova, G V; Rubtsova, M Yu; Egorov, A M; Grigorenko, V G; Yaminsky, I V

    2015-01-01

    Oligonucleotide microarrays are considered today to be one of the most efficient methods of gene diagnostics. The capability of atomic force microscopy (AFM) to characterize the three-dimensional morphology of single molecules on a surface allows one to use it as an effective tool for the 3D analysis of a microarray for the detection of nucleic acids. The high resolution of AFM offers ways to decrease the detection threshold of target DNA and increase the signal-to-noise ratio. In this work, we suggest an approach to the evaluation of the results of hybridization of gold nanoparticle-labeled nucleic acids on silicon microarrays based on an AFM analysis of the surface both in air and in liquid which takes into account of their three-dimensional structure. We suggest a quantitative measure of the hybridization results which is based on the fraction of the surface area occupied by the nanoparticles.

  20. Exploiting protected maleimides to modify oligonucleotides, peptides and peptide nucleic acids.

    Science.gov (United States)

    Paris, Clément; Brun, Omar; Pedroso, Enrique; Grandas, Anna

    2015-04-10

    This manuscript reviews the possibilities offered by 2,5-dimethylfuran-protected maleimides. Suitably derivatized building blocks incorporating the exo Diels-Alder cycloadduct can be introduced at any position of oligonucleotides, peptide nucleic acids, peptides and peptoids, making use of standard solid-phase procedures. Maleimide deprotection takes place upon heating, which can be followed by either Michael-type or Diels-Alder click conjugation reactions. However, the one-pot procedure in which maleimide deprotection and conjugation are simultaneously carried out provides the target conjugate more quickly and, more importantly, in better yield. This procedure is compatible with conjugates involving oligonucleotides, peptides and peptide nucleic acids. A variety of cyclic peptides and oligonucleotides can be obtained from peptide and oligonucleotide precursors incorporating protected maleimides and thiols.

  1. The 2010 Nucleic Acids Research Database Issue and online Database Collection: a community of data resources.

    Science.gov (United States)

    Cochrane, Guy R; Galperin, Michael Y

    2010-01-01

    The current issue of Nucleic Acids Research includes descriptions of 58 new and 73 updated data resources. The accompanying online Database Collection, available at http://www.oxfordjournals.org/nar/database/a/, now lists 1230 carefully selected databases covering various aspects of molecular and cell biology. While most data resource descriptions remain very brief, the issue includes several longer papers that highlight recent significant developments in such databases as Pfam, MetaCyc, UniProt, ELM and PDBe. The databases described in the Database Issue and Database Collection, however, are far more than a distinct set of resources; they form a network of connected data, concepts and shared technology. The full content of the Database Issue is available online at the Nucleic Acids Research web site (http://nar.oxfordjournals.org/).

  2. Single-Labeled Oligonucleotides Showing Fluorescence Changes upon Hybridization with Target Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Gil Tae Hwang

    2018-01-01

    Full Text Available Sequence-specific detection of nucleic acids has been intensively studied in the field of molecular diagnostics. In particular, the detection and analysis of single-nucleotide polymorphisms (SNPs is crucial for the identification of disease-causing genes and diagnosis of diseases. Sequence-specific hybridization probes, such as molecular beacons bearing the fluorophore and quencher at both ends of the stem, have been developed to enable DNA mutation detection. Interestingly, DNA mutations can be detected using fluorescently labeled oligonucleotide probes with only one fluorophore. This review summarizes recent research on single-labeled oligonucleotide probes that exhibit fluorescence changes after encountering target nucleic acids, such as guanine-quenching probes, cyanine-containing probes, probes containing a fluorophore-labeled base, and microenvironment-sensitive probes.

  3. Clarithromycin, trimethoprim, and penicillin and oxidative nucleic acid modifications in humans: randomised, controlled trials

    DEFF Research Database (Denmark)

    Larsen, Emil List; Cejvanovic, Vanja; Kjær, Laura Kofoed

    2017-01-01

    , phenoxymethylpenicillin (penicillin V), or placebo. Oxidative modifications were measured as 24-h urinary excretion of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and plasma levels of malondialdehyde before and after treatment as a measurement of DNA oxidation, RNA oxidation.......7% (95% CI: 5.8–37.6%), but did not influence urinary excretion of 8-oxoGuo. Penicillin V did not influence urinary excretion of 8-oxodG or 8-oxoGuo. None of the antibiotic drugs influenced plasma levels of malondialdehyde. Conclusion Clarithromycin significantly increases oxidative nucleic acid...... modifications. Increased oxidative modifications might explain some of clarithromycin's known adverse reactions. Trimethoprim significantly lowers DNA oxidation but not RNA oxidation. Penicillin V had no effect on oxidative nucleic acid modifications....

  4. Crystallographic studies of drug--nucleic acid crystalline complexes and their biological implications

    Energy Technology Data Exchange (ETDEWEB)

    Sobell, H.M.; Jain, S.C.; Sakore, T.D.; Reddy, B.S.; Bhandary, K.K.; Seshadri, T.P.

    1978-01-01

    It is now over a decade since Lerman proposed his intercalation hypothesis to explain the strong binding mode of the aminoacridines to DNA. A large body of evidence now supports this concept, and it has become increasingly apparent that (in addition to the aminoacridines) a large number of additional drugs and dyes bind to DNA by intercalation. The technique of molecular cocrystallization as developed in our laboratory has provided an opportunity to obtain detailed stereochemical information about interactions between many of these drugs and nucleic acid components and this has led to unifying structural concepts in understanding a large number of drug-DNA interactions. This paper will summarize the results of our crystallographic studies of drug-nucleic acid crystalline complexes done in recent years. It also discusses their broader biological implications in further detail.

  5. JAWS: Just Add Water System - A device for detection of nucleic acids in Martian ice caps

    DEFF Research Database (Denmark)

    Hansen, Anders J.; Willerslev, Eske; Mørk, Søren

    2002-01-01

    with a regulation of pH and salt concentrations e.g. the MOD systems and could be installed on a planetary probe melting its way down the Martian ice caps e.g. the NASA Cryobot. JAWS can be used for detection of remains of ancient life preserved in the Martian ice as well as for detection of contamination brought......The design of a device for nucleic acid detection in the Martian ice caps is presented; the Just Add Water System (JAWS). It is based on fiber-optic PNA (peptide nucleic acid) light up probe random microsphere universal array technology. JAWS is designed to be part of a larger system...

  6. DNA sequencing validation of Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acid tests.

    Science.gov (United States)

    Lee, Sin Hang; Vigliotti, Veronica S; Pappu, Suri

    2008-06-01

    DNA sequencing was used to confirm Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids in endocervical swab samples. DNA in residues of the samples with positive results by 2 commercial kits was subjected to nested polymerase chain reaction (PCR) amplification. The nested PCR amplicons were used as templates for direct automated DNA sequencing. A 40-base signature sequence was sufficient to achieve unequivocal validation of C trachomatis cryptic plasmid and gonococcal opa gene DNA. DNA with a signature sequence specific for C trachomatis was identified in all 14 samples and for N gonorrhoeae in all 13 samples with positive results by the commercial kits for these 2 microbes. In a low-prevalence population, PCR retesting of 289 samples with initial negative results by a non-nucleic acid amplification assay revealed 3 samples positive for C trachomatis and 2 samples positive for N gonorrhoeae that were missed by the commercial kit. DNA sequencing is a useful tool in validating molecular diagnostics.

  7. Exploiting Protected Maleimides to Modify Oligonucleotides, Peptides and Peptide Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Clément Paris

    2015-04-01

    Full Text Available This manuscript reviews the possibilities offered by 2,5-dimethylfuran-protected maleimides. Suitably derivatized building blocks incorporating the exo Diels-Alder cycloadduct can be introduced at any position of oligonucleotides, peptide nucleic acids, peptides and peptoids, making use of standard solid-phase procedures. Maleimide deprotection takes place upon heating, which can be followed by either Michael-type or Diels-Alder click conjugation reactions. However, the one-pot procedure in which maleimide deprotection and conjugation are simultaneously carried out provides the target conjugate more quickly and, more importantly, in better yield. This procedure is compatible with conjugates involving oligonucleotides, peptides and peptide nucleic acids. A variety of cyclic peptides and oligonucleotides can be obtained from peptide and oligonucleotide precursors incorporating protected maleimides and thiols.

  8. The "Jekyll and Hyde" Actions of Nucleic Acids on the Prion-like Aggregation of Proteins.

    Science.gov (United States)

    Silva, Jerson L; Cordeiro, Yraima

    2016-07-22

    Protein misfolding results in devastating degenerative diseases and cancer. Among the culprits involved in these illnesses are prions and prion-like proteins, which can propagate by converting normal proteins to the wrong conformation. For spongiform encephalopathies, a real prion can be transmitted among individuals. In other disorders, the bona fide prion characteristics are still under investigation. Besides inducing misfolding of native proteins, prions bind nucleic acids and other polyanions. Here, we discuss how nucleic acid binding might influence protein misfolding for both disease-related and benign, functional prions and why the line between bad and good amyloids might be more subtle than previously thought. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Nanomedicine-based combination anticancer therapy between nucleic acids and small-molecular drugs.

    Science.gov (United States)

    Huang, Wei; Chen, Liqing; Kang, Lin; Jin, Mingji; Sun, Ping; Xin, Xin; Gao, Zhonggao; Bae, You Han

    2017-06-01

    Anticancer therapy has always been a vital challenge for the development of nanomedicine. Repeated single therapeutic agent may lead to undesirable and severe side effects, unbearable toxicity and multidrug resistance due to complex nature of tumor. Nanomedicine-based combination anticancer therapy can synergistically improve antitumor outcomes through multiple-target therapy, decreasing the dose of each therapeutic agent and reducing side effects. There are versatile combinational anticancer strategies such as chemotherapeutic combination, nucleic acid-based co-delivery, intrinsic sensitive and extrinsic stimulus combinational patterns. Based on these combination strategies, various nanocarriers and drug delivery systems were engineered to carry out the efficient co-delivery of combined therapeutic agents for combination anticancer therapy. This review focused on illustrating nanomedicine-based combination anticancer therapy between nucleic acids and small-molecular drugs for synergistically improving anticancer efficacy. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Solution Preserves Nucleic Acids in Body-Fluid Specimens

    Science.gov (United States)

    Pierson, Duane L.; Stowe, Raymond P.

    2004-01-01

    A solution has been formulated to preserve deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in specimens of blood, saliva, and other bodily fluids. Specimens of this type are collected for diagnostic molecular pathology, which is becoming the method of choice for diagnosis of many diseases. The solution makes it possible to store such specimens at room temperature, without risk of decomposition, for subsequent analysis in a laboratory that could be remote from the sampling location. Thus, the solution could be a means to bring the benefits of diagnostic molecular pathology to geographic regions where refrigeration equipment and diagnostic laboratories are not available. The table lists the ingredients of the solution. The functions of the ingredients are the following: EDTA chelates divalent cations that are necessary cofactors for nuclease activity. In so doing, it functionally removes these cations and thereby retards the action of nucleases. EDTA also stabilizes the DNA helix. Tris serves as a buffering agent, which is needed because minor contaminants in an unbuffered solution can exert pronounced effects on pH and thereby cause spontaneous degradation of DNA. SDS is an ionic detergent that inhibits ribonuclease activity. SDS has been used in some lysis buffers and as a storage buffer for RNA after purification. The use of the solution is straightforward. For example, a sample of saliva is collected by placing a cotton roll around in the subject's mouth until it becomes saturated, then the cotton is placed in a collection tube. Next, 1.5 mL of the solution are injected directly into the cotton and the tube is capped for storage at room temperature. The effectiveness of the solution has been demonstrated in tests on specimens of saliva containing herpes simplex virus. In the tests, the viral DNA, as amplified by polymerase chain reaction, was detected even after storage for 120 days.

  11. Experimental Warming Decreases the Average Size and Nucleic Acid Content of Marine Bacterial Communities

    KAUST Repository

    Huete-Stauffer, Tamara M.

    2016-05-23

    Organism size reduction with increasing temperature has been suggested as a universal response to global warming. Since genome size is usually correlated to cell size, reduction of genome size in unicells could be a parallel outcome of warming at ecological and evolutionary time scales. In this study, the short-term response of cell size and nucleic acid content of coastal marine prokaryotic communities to temperature was studied over a full annual cycle at a NE Atlantic temperate site. We used flow cytometry and experimental warming incubations, spanning a 6°C range, to analyze the hypothesized reduction with temperature in the size of the widespread flow cytometric bacterial groups of high and low nucleic acid content (HNA and LNA bacteria, respectively). Our results showed decreases in size in response to experimental warming, which were more marked in 0.8 μm pre-filtered treatment rather than in the whole community treatment, thus excluding the role of protistan grazers in our findings. Interestingly, a significant effect of temperature on reducing the average nucleic acid content (NAC) of prokaryotic cells in the communities was also observed. Cell size and nucleic acid decrease with temperature were correlated, showing a common mean decrease of 0.4% per °C. The usually larger HNA bacteria consistently showed a greater reduction in cell and NAC compared with their LNA counterparts, especially during the spring phytoplankton bloom period associated to maximum bacterial growth rates in response to nutrient availability. Our results show that the already smallest planktonic microbes, yet with key roles in global biogeochemical cycling, are likely undergoing important structural shrinkage in response to rising temperatures.

  12. Nucleic acid molecules conferring enhanced ethanol tolerance and microorganisms having enhanced tolerance to ethanol

    Science.gov (United States)

    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.

  13. The 2016 database issue of Nucleic Acids Research and an updated molecular biology database collection

    Science.gov (United States)

    Rigden, Daniel J.; Fernández-Suárez, Xosé M.; Galperin, Michael Y.

    2016-01-01

    The 2016 Database Issue of Nucleic Acids Research starts with overviews of the resources provided by three major bioinformatics centers, the U.S. National Center for Biotechnology Information (NCBI), the European Bioinformatics Institute (EMBL-EBI) and Swiss Institute for Bioinformatics (SIB). Also included are descriptions of 62 new databases and updates on 95 databases that have been previously featured in NAR plus 17 previously described elsewhere. A number of papers in this issue deal with resources on nucleic acids, including various kinds of non-coding RNAs and their interactions, molecular dynamics simulations of nucleic acid structure, and two databases of super-enhancers. The protein database section features important updates on the EBI's Pfam, PDBe and PRIDE databases, as well as a variety of resources on pathways, metabolomics and metabolic modeling. This issue also includes updates on popular metagenomics resources, such as MG-RAST, EBI Metagenomics, and probeBASE, as well as a newly compiled Human Pan-Microbe Communities database. A significant fraction of the new and updated databases are dedicated to the genetic basis of disease, primarily cancer, and various aspects of drug research, including resources for patented drugs, their side effects, withdrawn drugs, and potential drug targets. A further six papers present updated databases of various antimicrobial and anticancer peptides. The entire Database Issue is freely available online on the Nucleic Acids Research website (http://nar.oxfordjournals.org/). The NAR online Molecular Biology Database Collection, http://www.oxfordjournals.org/nar/database/c/, has been updated with the addition of 88 new resources and removal of 23 obsolete websites, which brought the current listing to 1685 databases. PMID:26740669

  14. A measure of bending in nucleic acids structures applied to A-tract DNA

    Czech Academy of Sciences Publication Activity Database

    Lankaš, Filip; Špačková, Naďa; Moakher, M.; Enkhbayar, P.; Šponer, Jiří

    2010-01-01

    Roč. 38, č. 10 (2010), s. 3414-3422 ISSN 0305-1048 R&D Projects: GA MŠk(CZ) LC06030 Grant - others:GA MŠk(CZ) LC512 Program:LC Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702; CEZ:AV0Z40550506 Keywords : nucleic acids * DNA * molecular dynamics Subject RIV: BO - Biophysics Impact factor: 7.836, year: 2010

  15. Nucleic Acid Aptamers: An Emerging Tool for Biotechnology and Biomedical Sensing

    Directory of Open Access Journals (Sweden)

    Ti-Hsuan Ku

    2015-07-01

    Full Text Available Detection of small molecules or proteins of living cells provides an exceptional opportunity to study genetic variations and functions, cellular behaviors, and various diseases including cancer and microbial infections. Our aim in this review is to give an overview of selected research activities related to nucleic acid-based aptamer techniques that have been reported in the past two decades. Limitations of aptamers and possible approaches to overcome these limitations are also discussed.

  16. DNA nanotechnology for nucleic acid analysis: DX motif-based sensor.

    Science.gov (United States)

    Kolpashchikov, Dmitry M; Gerasimova, Yulia V; Khan, Mohammad S

    2011-11-25

    A light on the tiles: A sensor that fluoresces in the presence of specific nucleic acids was designed and characterized. The sensor uses a molecular beacon probe and three adaptor strands to form a five-stranded assembly, a DX-tile, with a specific analyte. This sensor is a highly selective and affordable tool for the real-time analysis of DNA and RNA. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Development and Application of Nucleic Acid Hybridization Techniques to Arbovirus Surveillance and Diagnosis.

    Science.gov (United States)

    1986-11-04

    presence of specific hybrids can then be detected inmunologically using anti-biotin antibodies followed by signal amplification with an enzyie...techniques for detection of virus nucleic acid N) species: In the IAC virus system , progress has been made in both in situ hybridization and detection of...washed, blocked, and the detection system applied. Detection steps included steptavidin , biotinylated poly-alkaline phosphatase, and NBT-BCIP

  18. Nucleic acid binding properties of allicin: spectroscopic analysis and estimation of anti-tumor potential.

    Science.gov (United States)

    Tyagi, Gunjan; Pradhan, Shrikant; Srivastava, Tapasya; Mehrotra, Ranjana

    2014-01-01

    Allicin has received much attention due to its anti-proliferative activity and not-well elucidated underlying mechanism of action. This work focuses towards determining the cellular toxicity of allicin and understanding its interaction with nucleic acid at molecular level. MTT assay was used to assess the cell viability of A549 lung cancer cells against allicin. Fourier transform infrared (FTIR) and UV-visible spectroscopy were used to study the binding parameters of nucleic acid-allicin interaction. Allicin inhibits the proliferation of cancer cells in a concentration dependent manner. FTIR spectroscopy exhibited that allicin binds preferentially to minor groove of DNA via thymine base. Analysis of tRNA allicin complex has also revealed that allicin binds primarily through nitrogenous bases. Some amount of external binding with phosphate backbone was also observed for both DNA and RNA. UV visible spectra of both DNA allicin and RNA allicin complexes showed hypochromic shift with an estimated binding constant of 1.2×10(4)M(-1) for DNA and 1.06×10(3)M(-1)for RNA binding. No major transition from the B-form of DNA and A-form of RNA is observed after their interaction with allicin. The results demonstrated that allicin treatment inhibited the proliferation of A549 cells in a dose-dependent manner. Biophysical outcomes are suggestive of base binding and helix contraction of nucleic acid structure upon binding with allicin. The results describe cytotoxic potential of allicin and its binding properties with cellular nucleic acid, which could be helpful in deciphering the complete mechanism of cell death exerted by allicin. © 2013.

  19. DBBP: database of binding pairs in protein-nucleic acid interactions

    OpenAIRE

    Park, Byungkyu; Kim, Hyungchan; Han, Kyungsook

    2014-01-01

    Background Interaction of proteins with other molecules plays an important role in many biological activities. As many structures of protein-DNA complexes and protein-RNA complexes have been determined in the past years, several databases have been constructed to provide structure data of the complexes. However, the information on the binding sites between proteins and nucleic acids is not readily available from the structure data since the data consists mostly of the three-dimensional coordi...

  20. NNDB: the nearest neighbor parameter database for predicting stability of nucleic acid secondary structure.

    Science.gov (United States)

    Turner, Douglas H; Mathews, David H

    2010-01-01

    The Nearest Neighbor Database (NNDB, http://rna.urmc.rochester.edu/NNDB) is a web-based resource for disseminating parameter sets for predicting nucleic acid secondary structure stabilities. For each set of parameters, the database includes the set of rules with descriptive text, sequence-dependent parameters in plain text and html, literature references to experiments and usage tutorials. The initial release covers parameters for predicting RNA folding free energy and enthalpy changes.

  1. NNDB: the nearest neighbor parameter database for predicting stability of nucleic acid secondary structure

    OpenAIRE

    Turner, Douglas H.; David H Mathews

    2009-01-01

    The Nearest Neighbor Database (NNDB, http://rna.urmc.rochester.edu/NNDB) is a web-based resource for disseminating parameter sets for predicting nucleic acid secondary structure stabilities. For each set of parameters, the database includes the set of rules with descriptive text, sequence-dependent parameters in plain text and html, literature references to experiments and usage tutorials. The initial release covers parameters for predicting RNA folding free energy and enthalpy changes.

  2. The 2014 Nucleic Acids Research Database Issue and an updated NAR online Molecular Biology Database Collection

    OpenAIRE

    Fernández-Suárez, Xosé M.; Rigden, Daniel J.; Galperin, Michael Y.

    2013-01-01

    The 2014 Nucleic Acids Research Database Issue includes descriptions of 58 new molecular biology databases and recent updates to 123 databases previously featured in NAR or other journals. For convenience, the issue is now divided into eight sections that reflect major subject categories. Among the highlights of this issue are six databases of the transcription factor binding sites in various organisms and updates on such popular databases as CAZy, Database of Genomic Variants (DGV), dbGaP, D...

  3. Nucleic Acid Aptamers Against Biotoxins: A New Paradigm Toward the Treatment and Diagnostic Approach

    DEFF Research Database (Denmark)

    Lauridsen, Lasse Holm; Veedu, Rakesh N.

    2012-01-01

    to combat these problems. Fully sequestered in vitro, aptamers eliminate the need for a living host. Furthermore, one of the key advantages of using aptamers instead of antibodies is that they can be selected against very weakly immunogenic and cytotoxic substances. In this review, we focus on nucleic acid...... aptamers developed against various biotoxins of plant, microorganism, or animal origin and show how these can be used in diagnostics (e.g., biosensors) and therapy....

  4. Buckyballs conjugated with nucleic acid sequences identifies microorganisms in live cell assays.

    Science.gov (United States)

    Cheng, Qingsu; Parvin, Bahram

    2017-11-09

    Rapid identification of bacteria can play an important role at the point of care, evaluating the health of the ecosystem, and discovering spatiotemporal distributions of a bacterial community. We introduce a method for rapid identification of bacteria in live cell assays based on cargo delivery of a nucleic acid sequence and demonstrate how a mixed culture can be differentiated using a simple microfluidic system. C60 Buckyballs are functionalized with nucleic acid sequences and a fluorescent reporter to show that a diversity of microorganisms can be detected and identified in live cell assays. The nucleic acid complexes include an RNA detector, targeting a species-specific sequence in the 16S rRNA, and a complementary DNA with an attached fluorescent reporter. As a result, each bacterium can be detected and visualized at a specific emission frequency through fluorescence microscopy. The C60 probe complexes can detect and identify a diversity of microorganisms that include gram-position and negative bacteria, yeast, and fungi. More specifically, nucleic-acid probes are designed to identify mixed cultures of Bacillus subtilis and Streptococcus sanguinis, or Bacillus subtilis and Pseudomonas aeruginosa. The efficiency, cross talk, and accuracy for the C60 probe complexes are reported. Finally, to demonstrate that mixed cultures can be separated, a microfluidic system is designed that connects a single source-well to multiple sinks wells, where chemo-attractants are placed in the sink wells. The microfluidic system allows for differentiating a mixed culture. The technology allows profiling of bacteria composition, at a very low cost, for field studies and point of care.

  5. Nucleic acids encoding modified human immunodeficiency virus type 1 (HIV-1) group M consensus envelope glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Haynes, Barton F [Durham, NC; Gao, Feng [Durham, NC; Korber, Bette T [Los Alamos, NM; Hahn, Beatrice H [Birmingham, AL; Shaw, George M [Birmingham, AL; Kothe, Denise [Birmingham, AL; Li, Ying Ying [Hoover, AL; Decker, Julie [Alabaster, AL; Liao, Hua-Xin [Chapel Hill, NC

    2011-12-06

    The present invention relates, in general, to an immunogen and, in particular, to an immunogen for inducing antibodies that neutralizes a wide spectrum of HIV primary isolates and/or to an immunogen that induces a T cell immune response. The invention also relates to a method of inducing anti-HIV antibodies, and/or to a method of inducing a T cell immune response, using such an immunogen. The invention further relates to nucleic acid sequences encoding the present immunogens.

  6. Cation-Anion Interactions within the Nucleic Acid Ion Atmosphere Revealed by Ion Counting.

    Science.gov (United States)

    Gebala, Magdalena; Giambaşu, George M; Lipfert, Jan; Bisaria, Namita; Bonilla, Steve; Li, Guangchao; York, Darrin M; Herschlag, Daniel

    2015-11-25

    The ion atmosphere is a critical structural, dynamic, and energetic component of nucleic acids that profoundly affects their interactions with proteins and ligands. Experimental methods that "count" the number of ions thermodynamically associated with the ion atmosphere allow dissection of energetic properties of the ion atmosphere, and thus provide direct comparison to theoretical results. Previous experiments have focused primarily on the cations that are attracted to nucleic acid polyanions, but have also showed that anions are excluded from the ion atmosphere. Herein, we have systematically explored the properties of anion exclusion, testing the zeroth-order model that anions of different identity are equally excluded due to electrostatic repulsion. Using a series of monovalent salts, we find, surprisingly, that the extent of anion exclusion and cation inclusion significantly depends on salt identity. The differences are prominent at higher concentrations and mirror trends in mean activity coefficients of the electrolyte solutions. Salts with lower activity coefficients exhibit greater accumulation of both cations and anions within the ion atmosphere, strongly suggesting that cation-anion correlation effects are present in the ion atmosphere and need to be accounted for to understand electrostatic interactions of nucleic acids. To test whether the effects of cation-anion correlations extend to nucleic acid kinetics and thermodynamics, we followed the folding of P4-P6, a domain of the Tetrahymena group I ribozyme, via single-molecule fluorescence resonance energy transfer in solutions with different salts. Solutions of identical concentration but lower activity gave slower and less favorable folding. Our results reveal hitherto unknown properties of the ion atmosphere and suggest possible roles of oriented ion pairs or anion-bridged cations in the ion atmosphere for electrolyte solutions of salts with reduced activity. Consideration of these new results leads to

  7. Cation–Anion Interactions within the Nucleic Acid Ion Atmosphere Revealed by Ion Counting

    Science.gov (United States)

    Gebala, Magdalena; Giambasu, George M.; Lipfert, Jan; Bisaria, Namita; Bonilla, Steve; Li, Guangchao; York, Darrin M.; Herschlag, Daniel

    2016-01-01

    The ion atmosphere is a critical structural, dynamic, and energetic component of nucleic acids that profoundly affects their interactions with proteins and ligands. Experimental methods that “count” the number of ions thermodynamically associated with the ion atmosphere allow dissection of energetic properties of the ion atmosphere, and thus provide direct comparison to theoretical results. Previous experiments have focused primarily on the cations that are attracted to nucleic acid polyanions, but have also showed that anions are excluded from the ion atmosphere. Herein, we have systematically explored the properties of anion exclusion, testing the zeroth-order model that anions of different identity are equally excluded due to electrostatic repulsion. Using a series of monovalent salts, we find, surprisingly, that the extent of anion exclusion and cation inclusion significantly depends on salt identity. The differences are prominent at higher concentrations and mirror trends in mean activity coefficients of the electrolyte solutions. Salts with lower activity coefficients exhibit greater accumulation of both cations and anions within the ion atmosphere, strongly suggesting that cation–anion correlation effects are present in the ion atmosphere and need to be accounted for to understand electrostatic interactions of nucleic acids. To test whether the effects of cation–anion correlations extend to nucleic acid kinetics and thermodynamics, we followed the folding of P4–P6, a domain of the Tetrahymena group I ribozyme, via single-molecule fluorescence resonance energy transfer in solutions with different salts. Solutions of identical concentration but lower activity gave slower and less favorable folding. Our results reveal hitherto unknown properties of the ion atmosphere and suggest possible roles of oriented ion pairs or anion-bridged cations in the ion atmosphere for electrolyte solutions of salts with reduced activity. Consideration of these new

  8. A mechanically strengthened polyacrylamide gel matrix fully compatible with electrophoresis of proteins and nucleic acids.

    Science.gov (United States)

    Pushparajan, Charlotte; Goswami, Shailesh K; McAdam, Christopher J; Hanton, Lyall R; Dearden, Peter K; Moratti, Stephen C; Cridge, Andrew G

    2017-11-10

    Polyacrylamide gel electrophoresis is a universal tool in a biochemist's toolkit for protein and nucleic acid separation and subsequent visualisation and analysis. The standard formulation of polyacrylamide gels consists of acrylamide (ACM) monomer crosslinked with bisacrylamide (MBA) which creates a gel with excellent sieving properties but which is mechanically fragile and prone to tearing during post-electrophoresis manipulations involved in visualisation and analysis. By adding a poly(ethylene oxide) macro-crosslinker to the standard gel formulation, we have created a tough gel matrix that can be used to fractionate proteins and nucleic acids by polyacrylamide gel electrophoresis. The protein and nucleic acid resolving capabilities and performance during staining and electroblotting of the tough gel matrix rivals that of conventional acrylamide/bisacrylamide gels. The tough gel matrix is resistant to tear and remarkably elastic, capable of stretching to over four times its original length before breaking, and represents a significant improvement over standard polyacrylamide gel formulations. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  9. The 2018 Nucleic Acids Research database issue and the online molecular biology database collection

    Science.gov (United States)

    Fernández, Xosé M

    2018-01-01

    Abstract The 2018 Nucleic Acids Research Database Issue contains 181 papers spanning molecular biology. Among them, 82 are new and 84 are updates describing resources that appeared in the Issue previously. The remaining 15 cover databases most recently published elsewhere. Databases in the area of nucleic acids include 3DIV for visualisation of data on genome 3D structure and RNArchitecture, a hierarchical classification of RNA families. Protein databases include the established SMART, ELM and MEROPS while GPCRdb and the newcomer STCRDab cover families of biomedical interest. In the area of metabolism, HMDB and Reactome both report new features while PULDB appears in NAR for the first time. This issue also contains reports on genomics resources including Ensembl, the UCSC Genome Browser and ENCODE. Update papers from the IUPHAR/BPS Guide to Pharmacology and DrugBank are highlights of the drug and drug target section while a number of proteomics databases including proteomicsDB are also covered. The entire Database Issue is freely available online on the Nucleic Acids Research website (https://academic.oup.com/nar). The NAR online Molecular Biology Database Collection has been updated, reviewing 138 entries, adding 88 new resources and eliminating 47 discontinued URLs, bringing the current total to 1737 databases. It is available at http://www.oxfordjournals.org/nar/database/c/. PMID:29316735

  10. Enzyme-Free Nucleic Acid Amplification Assay Using a Cellphone-Based Well Plate Fluorescence Reader.

    Science.gov (United States)

    Kim, Donghyuk; Wei, Qingshan; Kim, Dong Hyeok; Tseng, Derek; Zhang, Jingzi; Pan, Eric; Garner, Omai; Ozcan, Aydogan; Di Carlo, Dino

    2018-01-02

    Nucleic acids, DNA and RNA, provide important fingerprint information for various pathogens and have significant diagnostic value; however, improved approaches are urgently needed to enable rapid detection of nucleic acids in simple point-of-care formats with high sensitivity and specificity. Here, we present a system that utilizes a series of toehold-triggered hybridization/displacement reactions that are designed to convert a given amount of RNA molecules (i.e., the analyte) into an amplified amount of signaling molecules without any washing steps or thermocycling. Fluorescent probes for signal generation were designed to consume products of the catalytic reaction in order to push the equilibrium and enhance the assay fold amplification for improved sensitivity and reaction speed. The system of toehold-assisted reactions is also modeled to better understand its performance and capabilities, and we empirically demonstrate the success of this approach with two analytes of diagnostic importance, i.e., influenza viral RNA and a micro RNA (miR-31). We also show that the amplified signal permits using a compact and cost-effective smartphone-based fluorescence reader, an important requirement toward a nucleic-acid-based point-of-care diagnostic system.

  11. Development of fluorescent nanoparticle-labeled lateral flow assay for the detection of nucleic acids.

    Science.gov (United States)

    Wang, Yuhong; Nugen, Sam R

    2013-10-01

    The rapid, specific and sensitive detection of nucleic acids is of utmost importance for the identification of infectious agents, diagnosis and treatment of genetic diseases, and the detection of pathogens related to human health and safety. Here we report the development of a simple and sensitive nucleic acid sequence-based and Ru(bpy)3 (2+)-doped silica nanoparticle-labeled lateral flow assay which achieves low limit of detection by using fluorescencent nanoparticles. The detection of the synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, was utilized to demonstrate this assay. The 30 nm spherical Ru(bpy)3 (2+)-doped silica nanoparticles were prepared in aqueous medium by a novel method recently reported. The nanoparticles were modified by 3-glycidoxypropyl trimethoxysilane in order to conjugate to amine-capped oligonucleotide reporter probes. The fluorescent intensities of the fluorescent assays were quantified on a mictrotiter plate reader using a custom holder. The experimental results showed that the lateral flow fluorescent assay developed was more sensitive compared with the traditional colloidal gold test strips. The limit of detection for the fluorescent lateral flow assay developed is approximately 0.066 fmols as compared to approximately 15 fmols for the colloidal gold. The limit of detection can further be reduced about one order of magnitude when "dipstick" format was used.

  12. Biophysical and structural characterisation of nucleic acid complexes with modified cyclodextrins using circular dichroism.

    Science.gov (United States)

    O'Mahony, Aoife M; Cronin, Michael F; McMahon, Anthony; Evans, James C; Daly, Kathleen; Darcy, Raphael; O'Driscoll, Caitriona M

    2014-05-01

    Modified cyclodextrins (CDs) have shown great promise as non-viral gene and siRNA delivery vectors in a range of in vitro and in vivo studies. In the current study, structural and biophysical characterisation of selected CDs was carried out to enhance our understanding of their interaction with nucleic acids. The methods used for such characterisation were dynamic light scattering, zeta potential measurements and circular dichroism. Variations in the chemistries of individual CDs and in the type of formulation were shown to affect key properties of complexes such as size, surface charge and nucleic acid conformation. Furthermore, the effects of temperature and pH on the conformation of nucleic acids were investigated. pH studies were intended to mimic the conditions encountered by cationic complexes during endocytosis. Circular dichroism studies revealed that changes occurred in DNA and siRNA conformation upon complexation with CDs and when exposed to increasing temperature and decreasing pH. Overall, siRNA appeared to be more susceptible to conformational changes although complexation of siRNA with CDs tended to have a stabilising effect. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  13. Nucleic-Acid-Binding Chromophores as Efficient Indicators of Aptamer-Target Interactions

    Directory of Open Access Journals (Sweden)

    Kwabena Sarpong

    2012-01-01

    Full Text Available The binding affinity and specificity of nucleic acid aptamers have made them valuable candidates for use as sensors in diagnostic applications. In particular, chromophore-functionalized aptamers offer a relatively simple format for detection and quantification of target molecules. We describe the use of nucleic-acid-staining reagents as an effective tool for detecting and signaling aptamer-target interactions. Aptamers varying in size and structure and targeting a range of molecules have been used in conjunction with commercially available chromophores to indicate and quantify the presence of cognate targets with high sensitivity and selectivity. Our assay precludes the covalent modification of nucleic acids and relies on the differential fluorescence signal of chromophores when complexed with aptamers with or without their cognate target. We also evaluate factors that are critical for the stability of the complex between the aptamer and chromophore in presence or absence of target molecules. Our results indicate the possibility of controlling those factors to enhance the sensitivity of target detection by the aptamers used in such assays.

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

    Science.gov (United States)

    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

  15. A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs.

    Directory of Open Access Journals (Sweden)

    Zhichao Miao

    2015-12-01

    Full Text Available Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i the definition of nucleic acid binding sites; ii the training and test datasets; iii the algorithmic methods for the prediction strategies; iv the performance measures and v the distribution and availability of the prediction programs. Here we report a large-scale assessment of 19 web servers and 3 stand-alone programs on 41 datasets including more than 5000 proteins derived from 3D structures of protein-nucleic acid complexes. Well-defined binary assessment criteria (specificity, sensitivity, precision, accuracy… are applied. We found that i the tools have been greatly improved over the years; ii some of the approaches suffer from theoretical defects and there is still room for sorting out the essential mechanisms of binding; iii RNA binding and DNA binding appear to follow similar driving forces and iv dataset bias may exist in some methods.

  16. Mucosal Immunization with Liposome-Nucleic Acid Adjuvants Generates Effective Humoral and Cellular Immunity

    Science.gov (United States)

    Henderson, Angela; Propst, Katie; Kedl, Ross; Dow, Steven

    2012-01-01

    Development of effective new mucosal vaccine adjuvants has become a priority with the increase in emerging viral and bacterial pathogens. We previously reported that cationic liposomes complexed with non-coding plasmid DNA (CLDC) were effective parenteral vaccine adjuvants. However, little is known regarding the ability of liposome-nucleic acid complexes to function as mucosal vaccine adjuvants, or the nature of the mucosal immune responses elicited by mucosal liposome-nucleic acid adjuvants. To address these questions, antibody and T cell responses were assessed in mice following intranasal immunization with CLDC-adjuvanted vaccines. The effects of CLDC adjuvant on antigen uptake, trafficking, and cytokine responses in the airways and draining lymph nodes were also assessed. We found that mucosal immunization with CLDC-adjuvanted vaccines effectively generated potent mucosal IgA antibody responses, as well as systemic IgG responses. Notably, mucosal immunization with CLDC adjuvant was very effective in generating strong and sustained antigen-specific CD8+ T cell responses in the airways of mice. Mucosal administration of CLDC vaccines also induced efficient uptake of antigen by DCs within the mediastinal lymph nodes. Finally, a killed bacterial vaccine adjuvanted with CLDC induced significant protection from lethal pulmonary challenge with Burkholderia pseudomallei. These findings suggest that liposome-nucleic acid adjuvants represent a promising new class of mucosal adjuvants for non-replicating vaccines, with notable efficiency at eliciting both humoral and cellular immune responses following intranasal administration. PMID:21600950

  17. Comparing nucleic acid lateral flow and electrochemical genosensing for the simultaneous detection of foodborne pathogens.

    Science.gov (United States)

    Ben Aissa, A; Jara, J J; Sebastián, R M; Vallribera, A; Campoy, S; Pividori, M I

    2017-02-15

    Due to the increasing need of rapid tests for application in low resource settings, WHO summarized their ideal features under the acronym ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, Delivered to those who need it). In this work, two different platforms for the rapid and simultaneous testing of the foodborne pathogens E. coli O157:H7 and Salmonella enterica, in detail a nucleic acid lateral flow and an electrochemical magneto-genosensor are presented and compared in terms of their analytical performance. The DNA of the bacteria was amplified by polymerase chain reaction using a quadruple-tagging set of primers specific for E. coli eaeA (151bp) and Salmonella enterica yfiR (375bp) genes. During the amplification, the amplicons were labelled at the same time with biotin/digoxigenin or biotin/fluorescein tags, respectively. The nucleic acid lateral flow assay was based on the use of streptavidin gold nanoparticles for the labelling of the tagged amplicon from E. coli and Salmonella. The visual readout was achieved when the gold-modified amplicons were captured by the specific antibodies. The features of this approach are discussed and compared with an electrochemical magneto-genosensor. Although nucleic acid lateral flow showed higher limit of detection, this strategy was able to clearly distinguish positive and negative samples of both bacteria being considered as a rapid and promising detection tool for bacteria screening. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Rapid amplification/detection of nucleic acid targets utilizing a HDA/thin film biosensor.

    Science.gov (United States)

    Jenison, Robert; Jaeckel, Heidi; Klonoski, Joshua; Latorra, David; Wiens, Jacinta

    2014-08-07

    Thin film biosensors exploit a flat, optically coated silicon-based surface whereupon formation of nucleic acid hybrids are enzymatically transduced in a molecular thin film that can be detected by the unaided human eye under white light. While the limit of sensitivity for detection of nucleic acid targets is at sub-attomole levels (60 000 copies) many clinical specimens containing bacterial pathogens have much lower levels of analyte present. Herein, we describe a platform, termed HDA/thin film biosensor, which performs helicase-dependant nucleic acid amplification on a thin film biosensor surface to improve the limit of sensitivity to 10 copies of the mecA gene present in methicillin-resistant strains of Staphylococcus. As double-stranded DNA is unwound by helicase it was either bound by solution-phase DNA primers to be copied by DNA polymerase or hybridized to surface immobilized probe on the thin film biosensor surface to be detected. Herein, we show that amplification reactions on the thin film biosensor are equivalent to in standard thin wall tubes, with detection at the limit of sensitivity of the assay occurring after 30 minutes of incubation time. Further we validate the approach by detecting the presence of the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) from positive blood culture aliquots with high specificity (signal/noise ratio of 105).

  19. Analyzing and predicting the thermodynamic effects of the metabolite trehalose on nucleic acids.

    Science.gov (United States)

    Hart, Jonathan L; Harris, Zachary M; Testa, Stephen M

    2010-12-01

    There is a lot of interest in exactly how nucleic acid duplexes are affected by the addition of certain stabilizing and destabilizing metabolites. Unfortunately, the differences in reaction conditions between published reports often precludes a comparison of the results, effectively preventing a cohesive strategy for predicting additive effects on nucleic acid stability. This information is critically important for obtaining a fundamental understanding of how additives, including metabolites, alter DNA and RNA stability and structure. We now show that the destabilization of nucleic acids by the metabolite trehalose in standard optical melting buffer (20 mM sodium cacodylate, 1M NaCl, and 0.5 mM EDTA) differs from that of a common PCR buffer, and a simulated physiological buffer, with up to an 8°C melting temperature difference. We also demonstrate that the extent of DNA destabilization due to trehalose depends on DNA length and depends on percent GC content, at least for the primer-length duplexes studied here. Furthermore, we show that glucose (a monomer) is not quite as effective a destabilizer as trehalose (a dimer). The implications of these results related to trehalose-destabilization of DNA, related to conducting and analyzing DNA-additive experiments, and related to using this type of data for predictive purposes are discussed. © 2010 Wiley Periodicals, Inc.

  20. Comparison of femtosecond laser and continuous wave UV sources for protein-nucleic acid crosslinking.

    Science.gov (United States)

    Fecko, Christopher J; Munson, Katherine M; Saunders, Abbie; Sun, Guangxing; Begley, Tadhg P; Lis, John T; Webb, Watt W

    2007-01-01

    Crosslinking proteins to the nucleic acids they bind affords stable access to otherwise transient regulatory interactions. Photochemical crosslinking provides an attractive alternative to formaldehyde-based protocols, but irradiation with conventional UV sources typically yields inadequate product amounts. Crosslinking with pulsed UV lasers has been heralded as a revolutionary technique to increase photochemical yield, but this method had only been tested on a few protein-nucleic acid complexes. To test the generality of the yield enhancement, we have investigated the benefits of using approximately 150 fs UV pulses to crosslink TATA-binding protein, glucocorticoid receptor and heat shock factor to oligonucleotides in vitro. For these proteins, we find that the quantum yields (and saturating yields) for forming crosslinks using the high-peak intensity femtosecond laser do not improve on those obtained with low-intensity continuous wave (CW) UV sources. The photodamage to the oligonucleotides and proteins also has comparable quantum yields. Measurements of the photochemical reaction yields of several small molecules selected to model the crosslinking reactions also exhibit nearly linear dependences on UV intensity instead of the previously predicted quadratic dependence. Unfortunately, these results disprove earlier assertions that femtosecond pulsed laser sources provide significant advantages over CW radiation for protein-nucleic acid crosslinking.

  1. Evaluation of automated nucleic acid extraction methods for virus detection in a multicenter comparative trial.

    Science.gov (United States)

    Rasmussen, Thomas Bruun; Uttenthal, Ase; Hakhverdyan, Mikhayil; Belák, Sándor; Wakeley, Philip R; Reid, Scott M; Ebert, Katja; King, Donald P

    2009-01-01

    Five European veterinary laboratories participated in an exercise to compare the performance of nucleic acid extraction robots. Identical sets of coded samples were prepared using serial dilutions of bovine viral diarrhoea virus (BVDV) from serum and cell culture propagated material. Each laboratory extracted nucleic acid from this panel using available robotic equipment (12 separate instruments, comprising 8 different models), after which the processed samples were frozen and sent to a single laboratory for subsequent testing by real-time RT-PCR. In general, there was good concordance between the results obtained for the different automated extraction platforms. In particular, the limit of detection was identical for 9/12 and 8/12 best performing robots (using dilutions of BVDV infected-serum and cell culture material, respectively), which was similar to a manual extraction method used for comparison. The remaining equipment and protocols used were less sensitive, in an extreme case for serum, by a factor of 1000. There was no evidence for cross-contamination of RNA template in any of the negative samples included in these panels. These results are not intended to replace local optimisation and validation, but provide reassurance to laboratories to indicate that the best performing optimised nucleic acid extraction systems can have similar performance.

  2. The 2015 Nucleic Acids Research Database Issue and molecular biology database collection.

    Science.gov (United States)

    Galperin, Michael Y; Rigden, Daniel J; Fernández-Suárez, Xosé M

    2015-01-01

    The 2015 Nucleic Acids Research Database Issue contains 172 papers that include descriptions of 56 new molecular biology databases, and updates on 115 databases whose descriptions have been previously published in NAR or other journals. Following the classification that has been introduced last year in order to simplify navigation of the entire issue, these articles are divided into eight subject categories. This year's highlights include RNAcentral, an international community portal to various databases on noncoding RNA; ValidatorDB, a validation database for protein structures and their ligands; SASBDB, a primary repository for small-angle scattering data of various macromolecular complexes; MoonProt, a database of 'moonlighting' proteins, and two new databases of protein-protein and other macromolecular complexes, ComPPI and the Complex Portal. This issue also includes an unusually high number of cancer-related databases and other databases dedicated to genomic basics of disease and potential drugs and drug targets. The size of NAR online Molecular Biology Database Collection, http://www.oxfordjournals.org/nar/database/a/, remained approximately the same, following the addition of 74 new resources and removal of 77 obsolete web sites. The entire Database Issue is freely available online on the Nucleic Acids Research web site (http://nar.oxfordjournals.org/). Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  3. BIGNASim: a NoSQL database structure and analysis portal for nucleic acids simulation data.

    Science.gov (United States)

    Hospital, Adam; Andrio, Pau; Cugnasco, Cesare; Codo, Laia; Becerra, Yolanda; Dans, Pablo D; Battistini, Federica; Torres, Jordi; Goñi, Ramón; Orozco, Modesto; Gelpí, Josep Ll

    2016-01-04

    Molecular dynamics simulation (MD) is, just behind genomics, the bioinformatics tool that generates the largest amounts of data, and that is using the largest amount of CPU time in supercomputing centres. MD trajectories are obtained after months of calculations, analysed in situ, and in practice forgotten. Several projects to generate stable trajectory databases have been developed for proteins, but no equivalence exists in the nucleic acids world. We present here a novel database system to store MD trajectories and analyses of nucleic acids. The initial data set available consists mainly of the benchmark of the new molecular dynamics force-field, parmBSC1. It contains 156 simulations, with over 120 μs of total simulation time. A deposition protocol is available to accept the submission of new trajectory data. The database is based on the combination of two NoSQL engines, Cassandra for storing trajectories and MongoDB to store analysis results and simulation metadata. The analyses available include backbone geometries, helical analysis, NMR observables and a variety of mechanical analyses. Individual trajectories and combined meta-trajectories can be downloaded from the portal. The system is accessible through http://mmb.irbbarcelona.org/BIGNASim/. Supplementary Material is also available on-line at http://mmb.irbbarcelona.org/BIGNASim/SuppMaterial/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Contemporary nucleic acid-based molecular techniques for detection, identification, and characterization of Bifidobacterium.

    Science.gov (United States)

    Mianzhi, Yao; Shah, Nagendra P

    2017-03-24

    Bifidobacteria are one of the most important bacterial groups found in the gastrointestinal tract of humans. Medical and food industry researchers have focused on bifidobacteria because of their health-promoting properties. Researchers have historically relied on classic phenotypic approaches (culture and biochemical tests) for detection and identification of bifidobacteria. Those approaches still have values for the identification and detection of some bifidobacterial species, but they are often labor-intensive and time-consuming and can be problematic in differentiating closely related species. Rapid, accurate, and reliable methods for detection, identification, and characterization of bifidobacteria in a mixed bacterial population have become a major challenge. The advent of nucleic acid-based molecular techniques has significantly advanced isolation and detection of bifidobacteria. Diverse nucleic acid-based molecular techniques have been employed, including hybridization, target amplification, and fingerprinting. Certain techniques enable the detection, characterization, and identification at genus-, species-, and strains-levels, whereas others allow typing of species or strains of bifidobacteria. In this review, an overview of methodological principle, technique complexity, and application of various nucleic acid-based molecular techniques for detection, identification, and characterization of bifidobacteria is presented. Advantages and limitations of each technique are discussed, and significant findings based on particular techniques are also highlighted.

  5. New Fluorescent Nanoparticles for Ultrasensitive Detection of Nucleic Acids by Optical Methods.

    Science.gov (United States)

    Westergaard Mulberg, Mads; Taskova, Maria; Thomsen, Rasmus P; Okholm, Anders H; Kjems, Jørgen; Astakhova, Kira

    2017-08-17

    For decades the detection of nucleic acids and their interactions at low abundances has been a challenging task that has thus far been solved by enzymatic target amplification. In this work we aimed at developing efficient tools for amplification-free nucleic acid detection, which resulted in the synthesis of new fluorescent nanoparticles. Here, the fluorescent nanoparticles were made by simple and inexpensive radical emulsion polymerization of butyl acrylate in the presence of fluorescent dyes and additional functionalization reagents. This provided ultra-bright macrofluorophores of 9-84 nm mean diameter, modified with additional alkyne and amino groups for bioconjugation. By using click and NHS chemistries, the new nanoparticles were attached to target-specific DNA probes that were used in fluorimetry and fluorescence microscopy. Overall, these fluorescent nanoparticles and their oligonucleotide derivatives have higher photostability, brighter fluorescence and hence dramatically lower limits of target detection than the individual organic dyes. These properties make them useful in approaches directed towards ultrasensitive detection of nucleic acids, in particular for imaging and in vitro diagnostics of DNA. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Operating Cooperatively (OC sensor for highly specific recognition of nucleic acids.

    Directory of Open Access Journals (Sweden)

    Evan M Cornett

    Full Text Available Molecular Beacon (MB probes have been extensively used for nucleic acid analysis because of their ability to produce fluorescent signal in solution instantly after hybridization. The indirect binding of MB probe to a target analyte offers several advantages, including: improved genotyping accuracy and the possibility to analyse folded nucleic acids. Here we report on a new design for MB-based sensor, called 'Operating Cooperatively' (OC, which takes advantage of indirect binding of MB probe to a target analyte. The sensor consists of two unmodified DNA strands, which hybridize to a universal MB probe and a nucleic acid analyte to form a fluorescent complex. OC sensors were designed to analyze two human SNPs and E. coli 16S rRNA. High specificity of the approach was demonstrated by the detection of true analyte in over 100 times excess amount of single base substituted analytes. Taking into account the flexibility in the design and the simplicity in optimization, we conclude that OC sensors may become versatile and efficient tools for instant DNA and RNA analysis in homogeneous solution.

  7. Convenient synthesis and application of versatile nucleic acid lipid membrane anchors in the assembly and fusion of liposomes

    DEFF Research Database (Denmark)

    Ries, Oliver; Löffler, Philipp M. G.; Vogel, Stefan

    2015-01-01

    Hydrophobic moieties like lipid membrane anchors are highly demanded modifications for nucleic acid oligomers. Membrane-anchor modified oligonucleotides are applicable in biomedicine leading to new delivery strategies as well as in biophysical investigations towards assembly and fusion of liposom...

  8. Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain

    DEFF Research Database (Denmark)

    Koppelhus, Uffe; Shiraishi, Takehiko; Zachar, Vladimir

    2008-01-01

    Conjugation to cationic cell penetrating peptides (such as Tat, Penetratin, or oligo arginines) efficiently improves the cellular uptake of large hydrophilic molecules such as oligonucleotides and peptide nucleic acids, but the cellular uptake is predominantly via an unproductive endosomal pathwa...

  9. Medical devices; immunology and microbiology devices; classification of respiratory viral panel multiplex nucleic acid assay. Final rule.

    Science.gov (United States)

    2009-10-09

    The Food and Drug Administration (FDA) is announcing the classification of the respiratory viral panel multiplex nucleic acid assay into class II (special controls). The special controls that will apply to the device are three guidance documents entitled: "Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay," as applicable, "Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays," and as applicable,"Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.'' The agency classified the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device. Elsewhere in this issue of the Federal Register, FDA is announcing the availability of the guidance documents that will serve as the special controls for this device.

  10. Evidence for novel viruses by analysis of nucleic acids in virus-like particle fractions from Ambrosia psilostachya.

    Science.gov (United States)

    Melcher, Ulrich; Muthukumar, Vijay; Wiley, Graham B; Min, Byoung Eun; Palmer, Michael W; Verchot-Lubicz, Jeanmarie; Ali, Akhtar; Nelson, Richard S; Roe, Bruce A; Thapa, Vaskar; Pierce, Margaret L

    2008-09-01

    To test the hypothesis that many viruses remain to be discovered in plants, a procedure was developed to sequence nucleic acids cloned randomly from virus-like particle fractions of plant homogenates. As a test of the efficiency of the procedure we targeted Ambrosia psilostachya, western ragweed, plants growing at the Tallgrass Prairie Preserve of northeastern Oklahoma. Amplifiable nucleic acid was found in the fractions from six of twelve specimens and sequences were characterized from four of them. Evidence was obtained for the presence of viruses belonging to two families (Caulimoviridae, Flexiviridae). Multiple viral species were found in two of the four specimens and their level within the isolated nucleic acid population varied from less than 1-37%. None of the sequences were derived from reported sequences of known viruses. Thus, the analysis of nucleic acid from virus-like particles is a useful tool to expand our knowledge of the universe of viruses to non-cultivated species.

  11. A live gI/gE-deleted pseudorabies virus (PRV) protects weaned piglets against lethal variant PRV challenge.

    Science.gov (United States)

    Yin, Yue; Xu, Zhiwen; Liu, Xiaowan; Li, Ping; Yang, Fan; Zhao, Jun; Fan, Yi; Sun, Xiangang; Zhu, Ling

    2017-08-01

    Emerging pseudorabies virus (PRV) variant has led to frequent outbreaks of PRV infection among Bartha-K61-vaccinated swine population in Chinese swine farms and caused high mortality in pigs of all age since late 2011. Here, we generated a gE/gI-deleted PRV (rPRVXJ-delgI/gE-EGFP) based on PRV variant strain (PRV-XJ) through homologous DNA recombination. Compared to parental strain, rPRVXJ-delgI/gE-EGFP showed similar growth kinetics in vitro. Its safety and immunogenicity were evaluated in weaned piglets. Our results showed that piglets immunized with rPRVXJ-delgI/gE-EGFP did not exhibit any clinical symptoms, and a high level of gB-specific antibody was detected. After lethal challenge with variant PRV (PRV-FJ strain), all vaccinated piglets survived without showing any clinical symptoms except slight fever within 7 days post-challenge. In unvaccinated piglets, typical clinical symptoms of pseudorabies were observed, and the piglets were all died at 5 days post-challenge. These results indicated that a live rPRVXJ-delgI/gE-EGFP vaccine could be a maker vaccine candidate to control the currently epidemic pseudorabies in China.

  12. On-Chip, Amplification-Free Quantification of Nucleic Acid for Point-of-Care Diagnosis

    Science.gov (United States)

    Yen, Tony Minghung

    This dissertation demonstrates three physical device concepts to overcome limitations in point-of-care quantification of nucleic acids. Enabling sensitive, high throughput nucleic acid quantification on a chip, outside of hospital and centralized laboratory setting, is crucial for improving pathogen detection and cancer diagnosis and prognosis. Among existing platforms, microarray have the advantages of being amplification free, low instrument cost, and high throughput, but are generally less sensitive compared to sequencing and PCR assays. To bridge this performance gap, this dissertation presents theoretical and experimental progress to develop a platform nucleic acid quantification technology that is drastically more sensitive than current microarrays while compatible with microarray architecture. The first device concept explores on-chip nucleic acid enrichment by natural evaporation of nucleic acid solution droplet. Using a micro-patterned super-hydrophobic black silicon array device, evaporative enrichment is coupled with nano-liter droplet self-assembly workflow to produce a 50 aM concentration sensitivity, 6 orders of dynamic range, and rapid hybridization time at under 5 minutes. The second device concept focuses on improving target copy number sensitivity, instead of concentration sensitivity. A comprehensive microarray physical model taking into account of molecular transport, electrostatic intermolecular interactions, and reaction kinetics is considered to guide device optimization. Device pattern size and target copy number are optimized based on model prediction to achieve maximal hybridization efficiency. At a 100-mum pattern size, a quantum leap in detection limit of 570 copies is achieved using black silicon array device with self-assembled pico-liter droplet workflow. Despite its merits, evaporative enrichment on black silicon device suffers from coffee-ring effect at 100-mum pattern size, and thus not compatible with clinical patient samples. The

  13. Bio-Orthogonal Mediated Nucleic Acid Transfection of Cells via Cell Surface Engineering

    Science.gov (United States)

    2017-01-01

    The efficient delivery of foreign nucleic acids (transfection) into cells is a critical tool for fundamental biomedical research and a pillar of several biotechnology industries. There are currently three main strategies for transfection including reagent, instrument, and viral based methods. Each technology has significantly advanced cell transfection; however, reagent based methods have captured the majority of the transfection market due to their relatively low cost and ease of use. This general method relies on the efficient packaging of a reagent with nucleic acids to form a stable complex that is subsequently associated and delivered to cells via nonspecific electrostatic targeting. Reagent transfection methods generally use various polyamine cationic type molecules to condense with negatively charged nucleic acids into a highly positively charged complex, which is subsequently delivered to negatively charged cells in culture for association, internalization, release, and expression. Although this appears to be a straightforward procedure, there are several major issues including toxicity, low efficiency, sorting of viable transfected from nontransfected cells, and limited scope of transfectable cell types. Herein, we report a new strategy (SnapFect) for nucleic acid transfection to cells that does not rely on electrostatic interactions but instead uses an integrated approach combining bio-orthogonal liposome fusion, click chemistry, and cell surface engineering. We show that a target cell population is rapidly and efficiently engineered to present a bio-orthogonal functional group on its cell surface through nanoparticle liposome delivery and fusion. A complementary bio-orthogonal nucleic acid complex is then formed and delivered to which chemoselective click chemistry induced transfection occurs to the primed cell. This new strategy requires minimal time, steps, and reagents and leads to superior transfection results for a broad range of cell types

  14. Urinary markers of nucleic acid oxidation and long-term mortality of newly diagnosed type 2 diabetic patients

    DEFF Research Database (Denmark)

    Broedbaek, Kasper; Siersma, Volkert Dirk; Henriksen, Trine Maxel

    2011-01-01

    We analyzed data from a cohort of 1,381 newly diagnosed type 2 diabetic patients to test the hypothesis that urinary markers of nucleic acid oxidation are independent predictors of mortality.......We analyzed data from a cohort of 1,381 newly diagnosed type 2 diabetic patients to test the hypothesis that urinary markers of nucleic acid oxidation are independent predictors of mortality....

  15. Electromigration behavior of nucleic acids in capillary electrophoresis under pulsed-field conditions.

    Science.gov (United States)

    Li, Zhenqing; Liu, Chenchen; Dou, Xiaoming; Ni, Yi; Wang, Jiaxuan; Yamaguchi, Yoshinori

    2014-02-28

    We have presented a study focused on the migration pattern of double-stranded DNA (dsDNA) and RNA under pulsed field conditions. By calculating the dependence of nucleic acid mobility on its molecular size in a double logarithm plot, we found that (I) dsDNA molecules proceeded by a sigmoidal migration regime which was probably related to Ogston sieving, transition regime, and reptation model. Furthermore, the transition regime disappeared if DNA was resolved in a higher molecular mass HEC. (II) The migration pattern of RNA was relevant to the denaturant used for separation. When RNA was denatured by acetic acid, its mobility parabolically declined with its molecular size. The mobility was linearly decreased with the molecular size if urea was employed as denaturant. (III) RNA may migrate by Ogston, reptation without orientation mechanism when denatured by urea, whereas these two models were not suitable for RNA if denatured by acetic acid. Even though the electrophoretic conditions of PFCE were varied, the sigmoidal, linear, parabolic migration patterns could still be observed. (IV) Under certain modulation depth, the migration time (Tm) of acetic acid decreased with the increase of average separation voltage (Va), and when RNA denatured in 4.0M urea, Tm showed a linear correlation with Va. (V) The mobility of nucleic acids increased with the growth of artificial temperature in the capillary volume due to the decrease in the viscosity of the polymer. This is the first systematic and comparative research of high molecular mass nucleic acids in PFCE, which provides us deep insight into RNA and DNA migration behavior under pulsed electric field conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Germ-Cell-Specific Inflammasome Component NLRP14 Negatively Regulates Cytosolic Nucleic Acid Sensing to Promote Fertilization.

    Science.gov (United States)

    Abe, Takayuki; Lee, Albert; Sitharam, Ramaswami; Kesner, Jordan; Rabadan, Raul; Shapira, Sagi D

    2017-04-18

    Cytosolic sensing of nucleic acids initiates tightly regulated programs to limit infection. Oocyte fertilization represents a scenario wherein inappropriate responses to exogenous yet non-pathogen-derived nucleic acids would have negative consequences. We hypothesized that germ cells express negative regulators of nucleic acid sensing (NAS) in steady state and applied an integrated data-mining and functional genomics approach to identify a rheostat of DNA and RNA sensing-the inflammasome component NLRP14. We demonstrated that NLRP14 interacted physically with the nucleic acid sensing pathway and targeted TBK1 (TANK binding kinase 1) for ubiquitination and degradation. We further mapped domains in NLRP14 and TBK1 that mediated the inhibitory function. Finally, we identified a human nonsense germline variant associated with male sterility that results in loss of NLRP14 function and hyper-responsiveness to nucleic acids. The discovery points to a mechanism of nucleic acid sensing regulation that may be of particular importance in fertilization. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. MAZ-binding G4-decoy with locked nucleic acid and twisted intercalating nucleic acid modifications suppresses KRAS in pancreatic cancer cells and delays tumor growth in mice

    DEFF Research Database (Denmark)

    Cogoi, Susanna; Zorzet, Sonia; Rapozzi, Valentina

    2013-01-01

    transcription. To knockdown oncogenic KRAS in pancreatic cancer cells, we designed oligonucleotides that mimic one of the G-quadruplexes formed by NHE (G4-decoys). To increase their nuclease resistance, two locked nucleic acid (LNA) modifications were introduced at the 3'-end, whereas to enhance the folding...... cell growth and colony formation by activating apoptosis. We finally injected 2998 and control oligonucleotides 5153, 5154 (2 nmol/mouse) intratumorally in SCID mice bearing a Panc-1 xenograft. After three treatments, 2998 reduced tumor xenograft growth by 64% compared with control and increased......KRAS mutations are primary genetic lesions leading to pancreatic cancer. The promoter of human KRAS contains a nuclease-hypersensitive element (NHE) that can fold in G4-DNA structures binding to nuclear proteins, including MAZ (myc-associated zinc-finger). Here, we report that MAZ activates KRAS...

  18. Nucleic acid chaperons: a theory of an RNA-assisted protein folding

    Directory of Open Access Journals (Sweden)

    Biro Jan C

    2005-09-01

    Full Text Available Summary Background Proteins are assumed to contain all the information necessary for unambiguous folding (Anfinsen's principle. However, ab initio structure prediction is often not successful because the amino acid sequence itself is not sufficient to guide between endless folding possibilities. It seems to be a logical to try to find the "missing" information in nucleic acids, in the redundant codon base. Results mRNA energy dot plots and protein residue contact maps were found to be rather similar. The structure of mRNA is also conserved if the protein structure is conserved, even if the sequence similarity is low. These observations led me to suppose that some similarity might exist between nucleic acid and protein folding. I found that amino acid pairs, which are co-located in the protein structure, are preferentially coded by complementary codons. This codon complementarity is not perfect; it is suboptimal where the 1st and 3rd codon residues are complementary to each other in reverse orientation, while the 2nd codon letters may be, but are not necessarily, complementary. Conclusion Partial complementary coding of co-locating amino acids in protein structures suggests that mRNA assists in protein folding and functions not only as a template but even as a chaperon during translation. This function explains the role of wobble bases and answers the mystery of why we have a redundant codon base.

  19. The 2014 Nucleic Acids Research Database Issue and an updated NAR online Molecular Biology Database Collection.

    Science.gov (United States)

    Fernández-Suárez, Xosé M; Rigden, Daniel J; Galperin, Michael Y

    2014-01-01

    The 2014 Nucleic Acids Research Database Issue includes descriptions of 58 new molecular biology databases and recent updates to 123 databases previously featured in NAR or other journals. For convenience, the issue is now divided into eight sections that reflect major subject categories. Among the highlights of this issue are six databases of the transcription factor binding sites in various organisms and updates on such popular databases as CAZy, Database of Genomic Variants (DGV), dbGaP, DrugBank, KEGG, miRBase, Pfam, Reactome, SEED, TCDB and UniProt. There is a strong block of structural databases, which includes, among others, the new RNA Bricks database, updates on PDBe, PDBsum, ArchDB, Gene3D, ModBase, Nucleic Acid Database and the recently revived iPfam database. An update on the NCBI's MMDB describes VAST+, an improved tool for protein structure comparison. Two articles highlight the development of the Structural Classification of Proteins (SCOP) database: one describes SCOPe, which automates assignment of new structures to the existing SCOP hierarchy; the other one describes the first version of SCOP2, with its more flexible approach to classifying protein structures. This issue also includes a collection of articles on bacterial taxonomy and metagenomics, which includes updates on the List of Prokaryotic Names with Standing in Nomenclature (LPSN), Ribosomal Database Project (RDP), the Silva/LTP project and several new metagenomics resources. The NAR online Molecular Biology Database Collection, http://www.oxfordjournals.org/nar/database/c/, has been expanded to 1552 databases. The entire Database Issue is freely available online on the Nucleic Acids Research website (http://nar.oxfordjournals.org/).

  20. Terbium fluorescence as a sensitive, inexpensive probe for UV-induced damage in nucleic acids

    Energy Technology Data Exchange (ETDEWEB)

    El-Yazbi, Amira F.; Loppnow, Glen R., E-mail: glen.loppnow@ualberta.ca

    2013-07-05

    Graphical abstract: -- Highlights: •Simple, inexpensive, mix-and-read assay for positive detection of DNA damage. •Recognition of undamaged DNA via hybridization to a hairpin probe. •Terbium(III) fluorescence reports the amount of damage by binding to ssDNA. •Tb/hairpin is a highly selective and sensitive fluorescent probe for DNA damage. -- Abstract: Much effort has been focused on developing methods for detecting damaged nucleic acids. However, almost all of the proposed methods consist of multi-step procedures, are limited, require expensive instruments, or suffer from a high level of interferences. In this paper, we present a novel simple, inexpensive, mix-and-read assay that is generally applicable to nucleic acid damage and uses the enhanced luminescence due to energy transfer from nucleic acids to terbium(III) (Tb{sup 3+}). Single-stranded oligonucleotides greatly enhance the Tb{sup 3+} emission, but duplex DNA does not. With the use of a DNA hairpin probe complementary to the oligonucleotide of interest, the Tb{sup 3+}/hairpin probe is applied to detect ultraviolet (UV)-induced DNA damage. The hairpin probe hybridizes only with the undamaged DNA. However, the damaged DNA remains single-stranded and enhances the intrinsic fluorescence of Tb{sup 3+}, producing a detectable signal directly proportional to the amount of DNA damage. This allows the Tb{sup 3+}/hairpin probe to be used for sensitive quantification of UV-induced DNA damage. The Tb{sup 3+}/hairpin probe showed superior selectivity to DNA damage compared to conventional molecular beacons probes (MBs) and its sensitivity is more than 2.5 times higher than MBs with a limit of detection of 4.36 ± 1.2 nM. In addition, this probe is easier to synthesize and more than eight times cheaper than MBs, which makes its use recommended for high-throughput, quantitative analysis of DNA damage.

  1. The 2011 Nucleic Acids Research Database Issue and the online Molecular Biology Database Collection.

    Science.gov (United States)

    Galperin, Michael Y; Cochrane, Guy R

    2011-01-01

    The current 18th Database Issue of Nucleic Acids Research features descriptions of 96 new and 83 updated online databases covering various areas of molecular biology. It includes two editorials, one that discusses COMBREX, a new exciting project aimed at figuring out the functions of the 'conserved hypothetical' proteins, and one concerning BioDBcore, a proposed description of the 'minimal information about a biological database'. Papers from the members of the International Nucleotide Sequence Database collaboration (INSDC) describe each of the participating databases, DDBJ, ENA and GenBank, principles of data exchange within the collaboration, and the recently established Sequence Read Archive. A testament to the longevity of databases, this issue includes updates on the RNA modification database, Definition of Secondary Structure of Proteins (DSSP) and Homology-derived Secondary Structure of Proteins (HSSP) databases, which have not been featured here in >12 years. There is also a block of papers describing recent progress in protein structure databases, such as Protein DataBank (PDB), PDB in Europe (PDBe), CATH, SUPERFAMILY and others, as well as databases on protein structure modeling, protein-protein interactions and the organization of inter-protein contact sites. Other highlights include updates of the popular gene expression databases, GEO and ArrayExpress, several cancer gene databases and a detailed description of the UK PubMed Central project. The Nucleic Acids Research online Database Collection, available at: http://www.oxfordjournals.org/nar/database/a/, now lists 1330 carefully selected molecular biology databases. The full content of the Database Issue is freely available online at the Nucleic Acids Research web site (http://nar.oxfordjournals.org/).

  2. [Development of A synthetic positive control for the nucleic acid detection of mumps virus].

    Science.gov (United States)

    Cui, Ai-li; Jin, Li; Xu, Wen-bo

    2013-09-01

    To rapidly identify the cross-contamination problems caused by the positive control in the process of mumps virus nucleic acid detection, a new mumps virus RNA positive control was developed in this study. Using the same primers and reaction conditions, the cross-contamination problems caused by the positive control could be readily identified by comparing the fragments lengths of the PCR products between the positive control and the samples. This new RNA positive control of mumps virus can be widely used in the diagnosis and genotyping of mumps virus as a better laboratory quality control.

  3. Potent Antibacterial Antisense Peptide-Peptide Nucleic Acid Conjugates Against Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Ghosal, Anubrata; Nielsen, Peter E

    2012-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections in hospital settings, especially with immune compromised patients, and the increasing prevalence of multidrug resistant strains urges search for new drugs with novel mechanisms of action. In this study we introduce...... antisense peptide-peptide nucleic acid (PNA) conjugates as antibacterial agents against P. aeruginosa. We have designed and optimized antisense peptide-PNA conjugates targeting the translation initiation region of the ftsZ gene (an essential bacterial gene involved in cell division) or the acpP gene (an...... significantly reduced bacterial survival. These results open the possibility of development of antisense antibacterials for treatment of Pseudomonas infections....

  4. Rapid identification of Stenotrophomonas maltophilia by peptide nucleic acid fluorescence in situ hybridization.

    Science.gov (United States)

    Hansen, N; Rasmussen, A K I; Fiandaca, M J; Kragh, K N; Bjarnsholt, T; Høiby, N; Stender, H; Guardabassi, L

    2014-05-01

    The objective of this study was to develop a novel peptide nucleic acid (PNA) probe for Stenotrophomonas maltophilia identification by fluorescence in situ hybridization (FISH). The probe was evaluated using 33 human and veterinary clinical S. maltophilia isolates and 45 reference strains representing common bacterial species in the respiratory tract. The probe displayed 100% sensitivity and 100% specificity on pure cultures and allowed detection in sputum from cystic fibrosis patients. The detection limit was 10(4) CFU/mL in spiked tracheal aspirate and bronchoalveolar lavage from healthy horses. Altogether the study shows that this species-specific PNA FISH probe facilitates rapid detection of S. maltophilia in biological specimens.

  5. A comparison of nucleic acid content in Balantidium coli trophozoites from different isolates.

    Science.gov (United States)

    Skotarczak, B; Zieliński, R

    1997-01-01

    Cytophotometric assays were performed on Balantidium coli trophozoites isolated from 30 pigs affected by acute balantidiasis (Group I) and from 30 pigs with symptom-free balantidiasis (Group II). Trophozoites from cultures obtained from Group I and II pig isolates were assayed for comparison. Comparative cytophotometric studies on nucleic acids of B. coli trophozoites isolated from acute and symptomless balantidiasis-affected pigs as well as from in vitro cultured trophozoites showed differences which could have resulted from differences between populations in the trophozoans under investigation.

  6. Coupling Two Different Nucleic Acid Circuits in an Enzyme-Free Amplifier

    Directory of Open Access Journals (Sweden)

    Andrew D. Ellington

    2012-11-01

    Full Text Available DNA circuits have proven to be useful amplifiers for diagnostic applications, in part because of their modularity and programmability. In order to determine whether different circuits could be modularly stacked, we used a catalytic hairpin assembly (CHA circuit to initiate a hybridization chain reaction (HCR circuit. In response to an input nucleic acid sequence, the CHA reaction accumulates immobilized duplexes and HCR elongates these duplexes. With fluorescein as a reporter each of these processes yielded 10-fold signal amplification in a convenient 96-well format. The modular circuit connections also allowed the output reporter to be readily modified to a G-quadruplex-DNAzyme that yielded a fluorescent signal.

  7. Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA)

    DEFF Research Database (Denmark)

    Robaczewska, Magdalena; Narayan, Ramamurthy; Seigneres, Beatrice

    2005-01-01

    BACKGROUND/AIMS: Peptide nucleic acids (PNAs) appear as promising new antisense agents, that have not yet been examined as hepatitis B virus (HBV) inhibitors. Our aim was to study the ability of PNAs targeting the duck HBV (DHBV) encapsidation signal epsilon to inhibit reverse transcription (RT...... inhibitor than the PNA targeting only the bulge. Importantly, the inhibition was highly sequence-specific since double-mismatched PNA had no effect on the RT reaction. Moreover, in PDH the PNA coupled to Arg(7) cationic delivery peptide decreased DHBV replication. CONCLUSIONS: We provide the first evidence...

  8. Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer

    DEFF Research Database (Denmark)

    Pasternak, Anna; Hernandez, Frank J; Rasmussen, Lars Melholt

    2011-01-01

    A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA...... that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties...

  9. Convenient and Scalable Synthesis of Fmoc-Protected Peptide Nucleic Acid Backbone

    Directory of Open Access Journals (Sweden)

    Trevor A. Feagin

    2012-01-01

    Full Text Available The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA.

  10. Comparison of an automated nucleic acid extraction system with the column-based procedure

    OpenAIRE

    Frickmann, Hagen; Hinz, Rebecca; Hagen, Ralf Matthias

    2015-01-01

    Here, we assessed the extraction efficiency of a deployable bench-top nucleic acid extractor EZ1 in comparison to the column-based approach with complex sample matrices. A total of 48 EDTA blood samples and 81 stool samples were extracted by EZ1 automated extraction and the column-based QIAamp DNA Mini Kit. Blood sample extractions were assessed by two real-time malaria PCRs, while stool samples were analyzed by six multiplex real-time PCR assays targeting bacterial, viral, ...

  11. Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication

    DEFF Research Database (Denmark)

    Ndeboko, Benedicte; Ramamurthy, Narayan; Lemamy, Guy Joseph

    2017-01-01

    Peptide nucleic acids (PNAs) are potentially attractive antisense agents against hepatitis B virus (HBV), although poor cellular uptake limits their therapeutic application. In the duck HBV (DHBV) model, we evaluated different cell-penetrating peptides (CPPs) for delivery to hepatocytes of a PNA......-targeting hepadnaviral encapsidation signal (ε). This anti-ε PNA exhibited sequence-specific inhibition of DHBV RT in a cell-free system. Investigation of the best in vivo route of delivery of PNA conjugated to (D-Arg)8 (P1) showed that intraperitoneal injection to ducklings was ineffective, whereas intravenously (i...

  12. Isolation of nucleic acids and cultures from fossil ice and permafrost

    DEFF Research Database (Denmark)

    Willerslev, E.; Hansen, Anders J.; Poinar, H. N.

    2004-01-01

    and microbial nucleic acids obtained from ice- and permafrost cores from hundreds of thousands to millions of years old are not properly authenticated and the findings could be the result of contamination. Here, we discuss the processes that restrict the long-term survival of DNA and/or RNA molecules in ice...... and permafrost, and highlight sources of contamination that could result in false claims. Additionally, we present a set of precautions, controls and criteria to help ensure that future cultures and sequences are authentic. Udgivelsesdato: 2004 Mar...

  13. The use of atomic force microscopy for 3D analysis of nucleic acid hybridization on microarrays

    OpenAIRE

    Dubrovin, E.; Presnova, G.; Rubtsova, M.; Egorov, A.; Grigorenko, V.; Yaminsky, I.

    2015-01-01

    Oligonucleotide microarrays are considered today to be one of the most efficient methods of gene diagnostics. The capability of atomic force microscopy (AFM) to characterize the three-dimensional morphology of single molecules on a surface allows one to use it as an effective tool for the 3D analysis of a microarray for the detection of nucleic acids. The high resolution of AFM offers ways to decrease the detection threshold of target DNA and increase the signal-to-noise ratio. In this work, ...

  14. Application of Ammonium Persulfate for Selective Oxidation of Guanines for Nucleic Acid Sequencing

    Directory of Open Access Journals (Sweden)

    Yafen Wang

    2017-07-01

    Full Text Available Nucleic acids can be sequenced by a chemical procedure that partially damages the nucleotide positions at their base repetition. Many methods have been reported for the selective recognition of guanine. The accurate identification of guanine in both single and double regions of DNA and RNA remains a challenging task. Herein, we present a new, non-toxic and simple method for the selective recognition of guanine in both DNA and RNA sequences via ammonium persulfate modification. This strategy can be further successfully applied to the detection of 5-methylcytosine by using PCR.

  15. Nucleic acids encoding plant glutamine phenylpyruvate transaminase (GPT) and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

    2016-03-29

    Glutamine phenylpyruvate transaminase (GPT) proteins, nucleic acid molecules encoding GPT proteins, and uses thereof are disclosed. Provided herein are various GPT proteins and GPT gene coding sequences isolated from a number of plant species. As disclosed herein, GPT proteins share remarkable structural similarity within plant species, and are active in catalyzing the synthesis of 2-hydroxy-5-oxoproline (2-oxoglutaramate), a powerful signal metabolite which regulates the function of a large number of genes involved in the photosynthesis apparatus, carbon fixation and nitrogen metabolism.

  16. Using polyatomic primary ions to probe an amino acid and a nucleic base in water ice

    Energy Technology Data Exchange (ETDEWEB)

    Conlan, X.A. [Surface Analysis Research Centre, School of Chemical Engineering and Analytical Science, University of Manchester, P.O. Box 88, Manchester M60 1QD (United Kingdom)]. E-mail: x.conlan@postgrad.manchester.ac.uk; Biddulph, G.X. [Surface Analysis Research Centre, School of Chemical Engineering and Analytical Science, University of Manchester, P.O. Box 88, Manchester M60 1QD (United Kingdom)]. E-mail: G.Biddulph@postgrad.manchester.ac.uk; Lockyer, N.P. [Surface Analysis Research Centre, School of Chemical Engineering and Analytical Science, University of Manchester, P.O. Box 88, Manchester M60 1QD (United Kingdom); Vickerman, J.C. [Surface Analysis Research Centre, School of Chemical Engineering and Analytical Science, University of Manchester, P.O. Box 88, Manchester M60 1QD (United Kingdom)]. E-mail: John.Vickerman@manchester.ac.uk

    2006-07-30

    In this study on pure water ice, we show that protonated water species [H{sub 2}O] {sub n}H{sup +} are more prevalent than (H{sub 2}O) {sub n} {sup +} ions after bombardment by Au{sup +} monoatomic and Au{sub 3} {sup +} and C{sub 60} {sup +} polyatomic projectiles. This data also reveals significant differences in water cluster yields under bombardment by these three projectiles. The amino acid alanine and the nucleic base adenine in solution have been studied and have been shown to have an effect on the water cluster ion yields observed using an Au{sub 3} {sup +} ion beam.

  17. Bis-pyrene-modified unlocked nucleic acids: synthesis, hybridization studies, and fluorescent properties

    DEFF Research Database (Denmark)

    Perlíková, Pavla; Ejlersen, Maria; Langkjaer, Niels

    2014-01-01

    Efficient synthesis of a building block for the incorporation of a bis-pyrene-modified unlocked nucleic acid (UNA) into oligonucleotides (DNA*) was developed. The presence of bis-pyrene-modified UNA within a duplex leads to duplex destabilization that is more profound in DNA*/RNA and less distinc......)uracil:pyrene exciplex emission in the single-stranded form. Such fluorescent properties enable the application of bis-pyrene-modified UNA in the development of fluorescence probes for DNA/RNA detection and for detection of deletions at specific positions....

  18. Design of a New Type of Compact Chemical Heater for Isothermal Nucleic Acid Amplification.

    Directory of Open Access Journals (Sweden)

    Kamal G Shah

    Full Text Available Previous chemical heater designs for isothermal nucleic acid amplification have been based on solid-liquid phase transition, but using this approach, developers have identified design challenges en route to developing a low-cost, disposable device. Here, we demonstrate the feasibility of a new heater configuration suitable for isothermal amplification in which one reactant of an exothermic reaction is a liquid-gas phase-change material, thereby eliminating the need for a separate phase-change compartment. This design offers potentially enhanced performance and energy density compared to other chemical and electric heaters.

  19. Interaction of nucleic acid bases with single-walled carbon nanotube

    Science.gov (United States)

    Shukla, M. K.; Dubey, Madan; Zakar, Eugene; Namburu, Raju; Czyznikowska, Zaneta; Leszczynski, Jerzy

    2009-10-01

    Theoretical investigations at the M05-2X DFT level employing the 6-31G(d), 6-31G(d,p), 6-31+G(d,p) and cc-pVDZ basis sets show that all nucleic acid bases (NABs), guanine, adenine, cytosine, thymine and uracil form stable stacking complexes with the zigzag (7,0) single-walled carbon nanotube (SWCNT). The values of the BSSE corrected interaction energy suggested that among the bases guanine forms the most stable complex. The other bases generate complexes of similar stability with the considered SWCNT that are less stable than the guanine-SWCNT dimer.

  20. A Prebiotic Chemistry Experiment on the Adsorption of Nucleic Acids Bases onto a Natural Zeolite.

    Science.gov (United States)

    Anizelli, Pedro R; Baú, João Paulo T; Gomes, Frederico P; da Costa, Antonio Carlos S; Carneiro, Cristine E A; Zaia, Cássia Thaïs B V; Zaia, Dimas A M

    2015-09-01

    There are currently few mechanisms that can explain how nucleic acid bases were synthesized, concentrated from dilute solutions, and/or protected against degradation by UV radiation or hydrolysis on the prebiotic Earth. A natural zeolite exhibited the potential to adsorb adenine, cytosine, thymine, and uracil over a range of pH, with greater adsorption of adenine and cytosine at acidic pH. Adsorption of all nucleic acid bases was decreased in artificial seawater compared to water, likely due to cation complexation. Furthermore, adsorption of adenine appeared to protect natural zeolite from thermal degradation. The C=O groups from thymine, cytosine and uracil appeared to assist the dissolution of the mineral while the NH2 group from adenine had no effect. As shown by FT-IR spectroscopy, adenine interacted with a natural zeolite through the NH2 group, and cytosine through the C=O group. A pseudo-second-order model best described the kinetics of adenine adsorption, which occurred faster in artificial seawaters.

  1. Fluidic MEMS based Biosensor for the Detection of Nucleic acids by using Schizophyllan

    Science.gov (United States)

    Isoda, Takaaki; Hasegawa, Satoshi; Noguchi, Kazuhiro; Takamatsu, Kana; Itho, Fuyumi; Kimura, Taro; Sakurai, Kazuo; Shinkai, Seiji

    Recently, a fluid micro electro mechanical system (fluid MEMS), which is composed of a micro pump, mixer, valve, reactor, sensor, an electric circuit, on a chip, is being applied to a biotechnology or a medical analysis. Authors developed a micro sensor mounted on a chip to measure a concentration of one drop of solution (1nL-10μL) which contained nuclear acids. The form of the micro sensor mounted on the chip was a pair of Cu electrode which was a diameter of 2mm and was a thickness of 10μm. The sensing function was evaluated by the changes in a concentration of poly(x) (x=adenine, guanine, uracil, and cytosine) solution which was dropped on a micro sensor. To increase a recognization against nucleic acid, various adsorbent was added in a droplet. Especially, high recognition against the nucleic acid was observed with the adsorbent which modified the surface of polystyrene micro beads(10μm) using schizophyllan: a kind of polysaccharide. This biosensor recognized a small quantity of total-RNA and m-RNA extracted from the yeast.

  2. Nucleic acid distribution pattern in avian erythrocytes and mammalian lymphocytes: comparative studies by fluorescence microscopy and digital imaging analytical techniques.

    Science.gov (United States)

    Isitor, G N; Asgarali, Z; Pouching, K

    2008-12-01

    Nucleated erythrocytes of healthy domestic chicken and ducks, and lymphocytes of healthy Sprague Dawley rats were evaluated for nucleic acid distribution pattern, employing light and fluorescence microscopy procedures, as well as digital imaging analytical methods. The results demonstrate a unique organization of nuclear DNA of mature chicken and duck erythrocytes, as well as immature duck erythrocytes, as delineated spherical nuclear bodies that mostly corresponded with euchromatin zones of the cells in routine Wright-stain blood smears. The nuclear DNA of the rat lymphocytes, on the other hand, was observed as a more diffuse green fluorescing nuclear areas, with punctate variably-sized diffuse areas of RNA red fluorescence. RNA red color fluorescence was also evident in the narrow cytoplasm of the lymphocytes, especially in large lymphocytes, in comparison with the cytoplasm of the mature avian erythrocytes that completely lacked any nucleic acid fluorescence. Nuclear RNA fluorescence was lacking in the mature chicken erythrocytes, compared with those of the mature and immature duck erythrocytes as well as lymphocytes of both avian and rats blood. The significance of these findings lies in the establishment of normal benchmarks for the nuclear and cytoplasmic nucleic acid pattern in eukaryotic cells. These normal benchmarks become valuable in rapid diagnostic situations associated with pathologies, such as the presence of viral nuclear and cytoplasmic inclusion bodies that can alter the nucleic acid pattern of the host cells, and in conditions of cellular abnormal protein aggregations. Variability of cellular nucleic acid pattern can also aid in prognostic assessments of neoplastic conditions.

  3. A novel silicon based mags-biosensor for nucleic acid detection by magnetoelectronic transduction

    Directory of Open Access Journals (Sweden)

    Maria Eloisa Castagna

    2015-12-01

    Full Text Available We developed a novel silicon biosensor based on magnetoelectronic transduction (MAGS for nucleic acid detection. The mags-biosensor is a planar device composed by a primary micro-coil, and two secondary coils which produce a differential voltage due to the induced magnetic field. The presence of magnetic material over one of the secondary coils causes variations of induced magnetic field density that in turn results in a total output voltage different from zero. The voltage variation, therefore, is a measure of the amount of magnetic material present in the active zone. A device sensitivity of 5.1 mV/ng and a resolution of 0.008 ng have been observed. The biosensor also presents a micro-heater and a thermal sensor respectively to set and read-out the chip temperature: this aspect enables the device to be used for several biochemical applications that need temperature control and activation such for example nucleic acid amplification (real-time PCR, antigen- antibody detection (immune-assay and SNP detection.

  4. Functional nucleic acid-based hydrogels for bioanalytical and biomedical applications

    Science.gov (United States)

    Mo, Liuting; Lu, Chun-Hua; Fu, Ting

    2016-01-01

    Hydrogels are crosslinked hydrophilic polymers that can absorb a large amount of water. By their hydrophilic, biocompatible and highly tunable nature, hydrogels can be tailored for applications in bioanalysis and biomedicine. Of particular interest are DNA-based hydrogels owing to the unique features of nucleic acids. Since the discovery of DNA double helical structure, interest in DNA has expanded beyond its genetic role to applications in nanotechnology and materials science. In particular, DNA-based hydrogels present such remarkable features as stability, flexibility, precise programmability, stimuli-responsive DNA conformations, facile synthesis and modification. Moreover, functional nucleic acids (FNAs) have allowed the construction of hydrogels based on aptamers, DNAzymes, i-motif nanostructures, siRNAs and CpG oligodeoxynucleotides to provide additional molecular recognition, catalytic activities and therapeutic potential, making them key players in biological analysis and biomedical applications. To date, a variety of applications have been demonstrated with FNA-based hydrogels, including biosensing, environmental analysis, controlled drug release, cell adhesion and targeted cancer therapy. In this review, we focus on advances in the development of FNA-based hydrogels, which have fully incorporated both the unique features of FNAs and DNA-based hydrogels. We first introduce different strategies for constructing DNA-based hydrogels. Subsequently, various types of FNAs and the most recent developments of FNA-based hydrogels for bioanalytical and biomedical applications are described with some selected examples. Finally, the review provides an insight into the remaining challenges and future perspectives of FNA-based hydrogels. PMID:26758955

  5. Polysaccharide-free nucleic acids and proteins of Abelmoschus esculentus for versatile molecular studies.

    Science.gov (United States)

    Manoj-Kumar, A; Reddy, K N; Manjulatha, M; Blanco, L

    2012-01-01

    Abelmoschus esculentus (okra) is one of the polysaccharide rich crop plants. The polysaccharides interfere with nucleic acids and protein isolation thereby affecting the downstream molecular analysis. So, to understand the molecular systematics of okra, high quality DNA, RNA and proteins are essential. In this study we present a method for extracting genomic DNA, RNA and proteins from polysaccharide rich okra tissues. The conventional extraction procedures were integrated with purification treatments with pectinase, RNase and proteinase K, which improved the quality and quantity of DNA as well. Using SDS, additional washes with CIA and NaCl precipitation improved the RNA isolation both quantitatively and qualitatively. Finally, ammonium acetate mediated protein precipitation and re-solubilization increased the quality of total protein extracts from the okra leaves. All of the methods above not only eliminated the impurities but also improved the quality and quantity of nucleic acids and proteins. Further, we subjected these samples to versatile downstream molecular analyses such as restriction endonuclease digestion, RAPD, Southern, reverse transcription-PCR and Western analysis and were proved to be successful.

  6. Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label

    Directory of Open Access Journals (Sweden)

    Nattawut Yotapan

    2014-09-01

    Full Text Available DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV–vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA–DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes.

  7. An overlooked riddle of life's origins: energy-dependent nucleic acid unzipping.

    Science.gov (United States)

    Kovác, Ladislav; Nosek, Jozef; Tomáska, L'ubomír

    2003-01-01

    The imposing progress in understanding contemporary life forms on Earth and in manipulating them has not been matched by a comparable progress in understanding the origins of life. This paper argues that a crucial problem of unzipping of the double helix molecule of nucleic acid during its replication has been underrated, if not plainly overlooked, in the theories of life's origin and evolution. A model is presented of how evolution may have solved the problem in its early phase. Similar to several previous models, the model envisages the existence of a protocell, in which osmotic disbalance is being created by accumulation of synthetic products resulting in expansion and division of the protocell. Novel in the model is the presence in the protocell of a double-stranded nucleic acid, with each of its two strands being affixed by its 3'-terminus to the opposite sides of the membrane of a protocell. In the course of the protocell expansion, osmotic force is utilized to pull the two strands longitudinally in opposite directions, unzipping the helix and partitioning the strands between the two daughter protocells. The model is also being used as a background for arguments of why life need operate in cycles. Many formal models of life's origin and evolution have not taken into account the fact that logical possibility does not equal thermodynamic feasibility. A system of self-replication has to consist of both replicators and replicants.

  8. Label-free potentiometry for detecting DNA hybridization using peptide nucleic acid and DNA probes.

    Science.gov (United States)

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-02-07

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  9. Nucleic acid-based vaccines targeting respiratory syncytial virus: Delivering the goods.

    Science.gov (United States)

    Smith, Trevor R F; Schultheis, Katherine; Broderick, Kate E

    2017-11-02

    Respiratory syncytial virus (RSV) is a massive medical burden on a global scale. Infants, children and the elderly represent the vulnerable populations. Currently there is no approved vaccine to protect against the disease. Vaccine development has been hindered by several factors including vaccine enhanced disease (VED) associated with formalin-inactivated RSV vaccines, inability of target populations to raise protective immune responses after vaccination or natural viral infection, and a lack of consensus concerning the most appropriate virus-associated target antigen. However, with recent advances in the molecular understanding of the virus, and design of highly characterized vaccines with enhanced immunogenicity there is new belief a RSV vaccine is possible. One promising approach is nucleic acid-based vaccinology. Both DNA and mRNA RSV vaccines are showing promising results in clinically relevant animal models, supporting their transition into humans. Here we will discuss this strategy to target RSV, and the ongoing studies to advance the nucleic acid vaccine platform as a viable option to protect vulnerable populations from this important disease.

  10. ProtNA-ASA: Protein-nucleic acid structural database with information on accessible surface area

    Science.gov (United States)

    Tkachenko, M. Y.; Boryskina, O. P.; Shestopalova, A. V.; Tolstorukov, M. Y.

    The article describes a new database (ProtNA-ASA), which combines the data on conformational parameters of nucleic acids and calculations of the accessible surface area (ASA) of nucleic acid atoms in protein-DNA/RNA complexes. As for October 2008, the database contains 214 DNA-protein and 28 RNA-protein non-homologous complexes. The database provides structural parameters that describe local geometry of base pairs and base-pair steps as well as backbone torsion angles. Additionally, total ASA of DNA/RNA atoms and the accessible area of atoms in the minor and major grooves are calculated. ProtNA-ASA database facilitates studying the relationship between the DNA/RNA conformation and availability of atoms for contact with proteins either in major or in minor groove for different nucleotides. Such an analysis is important for understanding the principles of molecular recognition including indirect sequence readout. The database is publicly available for use at http://www.protna.bio-page.org.

  11. The 2012 Nucleic Acids Research Database Issue and the online Molecular Biology Database Collection.

    Science.gov (United States)

    Galperin, Michael Y; Fernández-Suárez, Xosé M

    2012-01-01

    The 19th annual Database Issue of Nucleic Acids Research features descriptions of 92 new online databases covering various areas of molecular biology and 100 papers describing recent updates to the databases previously described in NAR and other journals. The highlights of this issue include, among others, a description of neXtProt, a knowledgebase on human proteins; a detailed explanation of the principles behind the NCBI Taxonomy Database; NCBI and EBI papers on the recently launched BioSample databases that store sample information for a variety of database resources; descriptions of the recent developments in the Gene Ontology and UniProt Gene Ontology Annotation projects; updates on Pfam, SMART and InterPro domain databases; update papers on KEGG and TAIR, two universally acclaimed databases that face an uncertain future; and a separate section with 10 wiki-based databases, introduced in an accompanying editorial. The NAR online Molecular Biology Database Collection, available at http://www.oxfordjournals.org/nar/database/a/, has been updated and now lists 1380 databases. Brief machine-readable descriptions of the databases featured in this issue, according to the BioDBcore standards, will be provided at the http://biosharing.org/biodbcore web site. The full content of the Database Issue is freely available online on the Nucleic Acids Research web site (http://nar.oxfordjournals.org/).

  12. The 2013 Nucleic Acids Research Database Issue and the online molecular biology database collection.

    Science.gov (United States)

    Fernández-Suárez, Xosé M; Galperin, Michael Y

    2013-01-01

    The 20th annual Database Issue of Nucleic Acids Research includes 176 articles, half of which describe new online molecular biology databases and the other half provide updates on the databases previously featured in NAR and other journals. This year's highlights include two databases of DNA repeat elements; several databases of transcriptional factors and transcriptional factor-binding sites; databases on various aspects of protein structure and protein-protein interactions; databases for metagenomic and rRNA sequence analysis; and four databases specifically dedicated to Escherichia coli. The increased emphasis on using the genome data to improve human health is reflected in the development of the databases of genomic structural variation (NCBI's dbVar and EBI's DGVa), the NIH Genetic Testing Registry and several other databases centered on the genetic basis of human disease, potential drugs, their targets and the mechanisms of protein-ligand binding. Two new databases present genomic and RNAseq data for monkeys, providing wealth of data on our closest relatives for comparative genomics purposes. The NAR online Molecular Biology Database Collection, available at http://www.oxfordjournals.org/nar/database/a/, has been updated and currently lists 1512 online databases. The full content of the Database Issue is freely available online on the Nucleic Acids Research website (http://nar.oxfordjournals.org/).

  13. Urinary markers of nucleic acid oxidation in Danish overweight/obese children and youths

    DEFF Research Database (Denmark)

    Kloppenborg, Julie Tonsgaard; Fonvig, Cilius Esman; Johannesen, Jesper

    2016-01-01

    study we investigated the relationships between urinary markers of nucleic acid oxidation concentrations and the degree of obesity and glucose metabolism in overweight compared to lean children. 42 (24 girls) and 35 lean (19 girls) children and adolescents were recruited from the Registry of the Danish...... or glucose metabolism in lean and obese children. However, sub-analyses adjusted for age, sex and the degree of obesity showed positive associations between the two hour glucose (2 h glucose) and the urinary markers 8-oxoGuo (p=0.02, r(2)= 0.63) and 8-oxodG (p=0.046, r(2)= 0.48) and between the insulinogenic...... index and 8-oxoGuo (p=0.03, r(2)=0.60) in the 12 obese children exhibiting impaired glucose tolerance. Excretion of the urinary markers of nucleic acid oxidation and the degree of obesity or the glucose metabolism were not associated in this study. Nevertheless, obese children with impaired glucose...

  14. Quantitative reverse transcription strand displacement amplification: quantitation of nucleic acids using an isothermal amplification technique.

    Science.gov (United States)

    Nycz, C M; Dean, C H; Haaland, P D; Spargo, C A; Walker, G T

    1998-06-01

    Recent advances in nucleic acid amplification techniques have allowed for quantitation of viral nucleic acid levels in clinical specimens. The most prevalent testing is carried out for HIV viral load. Strand displacement amplification (SDA) is an isothermal DNA amplification system utilizing a restriction enzyme and a DNA polymerase with strand displacement properties. SDA was adapted for quantitative RNA amplification (QRT-SDA) of an HIV gag sequence by including AMV reverse transcriptase, a quantitative control sequence, and 32P-labeled detector oligonucleotides for the HIV and the control sequences. We have also improved the amplification efficiency by including the single-strand binding protein from gene 32 of T4 bacteriophage (T4gp32) to enhance strand displacement replication. In a preliminary analytical demonstration of the technique, RT-SDA was quantitative to within twofold over a range of 500-500,000 transcripts that were generated from a plasmid bearing an HIV gag sequence. QRT-SDA potentially represents a convenient alternative for viral load testing in a clinical setting. Copyright 1998 Academic Press.

  15. Limiting the level of tertiary amines on polyamines leads to biocompatible nucleic acid vectors.

    Science.gov (United States)

    Simão Carlos, Margarida Isabel; Zheng, Kai; Garrett, Natalie; Arifin, Natrah; Workman, David G; Kubajewska, Ilona; Halwani, Abdulrahman A; Moger, Julian; Zhang, Qi; Schätzlein, Andreas G; Uchegbu, Ijeoma F

    2017-06-30

    We have designed an efficient, synthetic nucleic acid vector, which is relatively non-toxic. [N-(2-ethylamino)-6-O-glycolchitosan - EAGC] polymers were 10-50 fold less toxic than Lipofectamine 2000, able to complex DNA, mRNA and siRNA into positively charged (zeta potential=+40 - 50mV), 50-450nm nanoparticles. The level of tertiary amine N-2-ethylamino substitution (DStert) was inversely proportional to the IC50 of the EAGC polymers in the A431 cell line: IC50=6.18DStert(-0.9), r(2)=0.9991. EAGC polyplexes were stable against a heparin challenge, able to protect the nucleic acids from nuclease degradation and achieve levels of transfection comparable to Lipofectamine 2000 formulations. The relative biocompatibility of the vector allowed 10 fold higher doses of DNA (1μg compared to 0.1μg per well with Lipofectamine 2000) and siRNA (10.7μg per well vs 1.3μg with Lipofectamine 2000) to be applied to cells, when compared to Lipofectamine 2000. Finally intranasal application of EAGC - siRNA complexes resulted in siRNA transfer to the neurons of the olfactory bulb. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. lncRNATargets: A platform for lncRNA target prediction based on nucleic acid thermodynamics.

    Science.gov (United States)

    Hu, Ruifeng; Sun, Xiaobo

    2016-08-01

    Many studies have supported that long noncoding RNAs (lncRNAs) perform various functions in various critical biological processes. Advanced experimental and computational technologies allow access to more information on lncRNAs. Determining the functions and action mechanisms of these RNAs on a large scale is urgently needed. We provided lncRNATargets, which is a web-based platform for lncRNA target prediction based on nucleic acid thermodynamics. The nearest-neighbor (NN) model was used to calculate binging-free energy. The main principle of NN model for nucleic acid assumes that identity and orientation of neighbor base pairs determine stability of a given base pair. lncRNATargets features the following options: setting of a specific temperature that allow use not only for human but also for other animals or plants; processing all lncRNAs in high throughput without RNA size limitation that is superior to any other existing tool; and web-based, user-friendly interface, and colored result displays that allow easy access for nonskilled computer operators and provide better understanding of results. This technique could provide accurate calculation on the binding-free energy of lncRNA-target dimers to predict if these structures are well targeted together. lncRNATargets provides high accuracy calculations, and this user-friendly program is available for free at http://www.herbbol.org:8001/lrt/ .

  17. Polymorphic Nucleic Acid Binding of Bioactive Isoquinoline Alkaloids and Their Role in Cancer

    Directory of Open Access Journals (Sweden)

    Motilal Maiti

    2010-01-01

    Full Text Available Bioactive alkaloids occupy an important position in applied chemistry and play an indispensable role in medicinal chemistry. Amongst them, isoquinoline alkaloids like berberine, palmatine and coralyne of protoberberine group, sanguinarine of the benzophenanthridine group, and their derivatives represent an important class of molecules for their broad range of clinical and pharmacological utility. In view of their extensive occurrence in various plant species and significantly low toxicities, prospective development and use of these alkaloids as effective anticancer agents are matters of great current interest. This review has focused on the interaction of these alkaloids with polymorphic nucleic acid structures (B-form, A-form, Z-form, HL-form, triple helical form, quadruplex form and their topoisomerase inhibitory activity reported by several research groups using various biophysical techniques like spectrophotometry, spectrofluorimetry, thermal melting, circular dichroism, NMR spectroscopy, electrospray ionization mass spectroscopy, viscosity, isothermal titration calorimetry, differential scanning calorimetry, molecular modeling studies, and so forth, to elucidate their mode and mechanism of action for structure-activity relationships. The DNA binding of the planar sanguinarine and coralyne are found to be stronger and thermodynamically more favoured compared to the buckled structure of berberine and palmatine and correlate well with the intercalative mechanism of sanguinarine and coralyne and the partial intercalation by berberine and palmatine. Nucleic acid binding properties are also interpreted in relation to their anticancer activity.

  18. Influenza and respiratory syncytial virus detection in clinical specimens without nucleic acid extraction using FOCUS direct disc assay is substantially equivalent to the traditional methods and the FOCUS nucleic acid extraction-dependent RT-PCR assay.

    Science.gov (United States)

    Selvaraju, Suresh B; Tierney, David; Leber, Amy L; Patel, Anami; Earley, Amalia K; Jaiswal, Dipeshkumar; Menegus, Marilyn A

    2014-03-01

    In this study, we evaluated FOCUS diagnostic's Flu A/B & RSV direct kit (Direct Disc assay), designed to detect influenza (FLU) and respiratory syncytial viruses (RSV) directly in clinical specimens without nucleic acid extraction. This novel 'sample-to-answer', nucleic acid extraction-independent assay uses a unique disc to process, amplify, and detect viral targets in up to 8 specimens at a time. The performance of this assay for detecting FLU and RSV viruses was compared to the traditional methods (culture and/or direct florescent antibody testing) using 945 nasopharyngeal swab specimens. In addition, a total of 150 consecutive clinical specimens positive for FLU (FLU A=50, FLU B=50) or RSV (n=50) were tested in parallel using the novel Direct Disc assay and FOCUS diagnostic's nucleic acid extraction-dependent assay to assess their relative performance. Compared to the traditional methods, the overall (prospective+retrospective) positive/negative percent agreement was determined to be 96.6%/98.1% for FLU A, 98.4%/99.9% for FLU B, and 99.3%/98.8% for RSV. Compared to the nucleic acid extraction-dependent assay, the positive percent agreement was 90% (n=45/50) for FLU A, 92% (n=46/50) for FLU B, and 98% (n=49/50) for RSV. Overall, the Direct Disc assay showed good agreement with both traditional methods and nucleic acid extraction-dependent assay. Although we encountered some failures compared to the nucleic acid extraction-dependent assay, these limitations must be balanced against the substantial advantages of the extraction-free nature of this assay and rapid turnaround time. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

    DEFF Research Database (Denmark)

    Fontenete, Sílvia; Joana, Barros; Pedro, Madureira

    2015-01-01

    acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect...... of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is performed with different types of FISH probes directly...... in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears...

  20. Cross-coupling reactions of nucleoside triphosphates followed by polymerase incorporation. Construction and applications of base-functionalized nucleic acids.

    Science.gov (United States)

    Hocek, Michal; Fojta, Miroslav

    2008-07-07

    Construction of functionalized nucleic acids (DNA or RNA) via polymerase incorporation of modified nucleoside triphosphates is reviewed and selected applications of the modified nucleic acids are highlighted. The classical multistep approach for the synthesis of modified NTPs by triphosphorylation of modified nucleosides is compared to the novel approach consisting of direct aqueous cross-coupling reactions of unprotected halogenated nucleoside triphosphates. The combination of cross-coupling of NTPs with polymerase incorporation gives an efficient and straightforward two-step synthesis of modified nucleic acids. Primer extension using biotinylated templates followed by separation using streptavidine-coated magnetic beads and DNA duplex denaturation is used for preparation of modified single stranded oligonucleotides. Examples of using this approach for electrochemical DNA labelling and bioanalytical applications are given.

  1. A Survey of Advancements in Nucleic Acid-based Logic Gates and Computing for Applications in Biotechnology and biomedicine

    Science.gov (United States)

    Wu, Cuichen; Wan, Shuo; Hou, Weijia; Zhang, Liqin; Xu, Jiehua; Cui, Cheng; Wang, Yanyue; Hu, Jun

    2015-01-01

    Nucleic acid-based logic devices were first introduced in 1994. Since then, science has seen the emergence of new logic systems for mimicking mathematical functions, diagnosing disease and even imitating biological systems. The unique features of nucleic acids, such as facile and high-throughput synthesis, Watson-Crick complementary base pairing, and predictable structures, together with the aid of programming design, have led to the widespread applications of nucleic acids (NA) for logic gating and computing in biotechnology and biomedicine. In this feature article, the development of in vitro NA logic systems will be discussed, as well as the expansion of such systems using various input molecules for potential cellular, or even in vivo, applications. PMID:25597946

  2. Synthetic LNA/DNA nano-scaffolds for highly efficient diagnostics of nucleic acids and autoimmune antibodies

    DEFF Research Database (Denmark)

    Astakhova, Irina Kira

    2014-01-01

    of the monoclonal human autoantibody is achieved. It makes the novel "clickable" LNA/DNA complexes a very promising tool in molecular diagnostics of both nucleic acids and autoantibodies against DNA. The latter are produced under several autoimmune conditions including antiphospholipide syndrome and systemic lupus......Herein novel fluorescent oligonucleotides for homogeneous (all-in-solution) detection of nucleic acids and autoimmune antibodies (autoantibodies) are described. The probes are prepared by highly efficient copper-catalyzed click chemistry between novel alkyne-modified locked nucleic acid (LNA......) strands and a series of fluorescent azides. The multiply labeled fluorescent LNA/DNA probes prepared herein generally display high binding affinity to complementary DNA/RNA, high quantum yields and, hence, high fluorescence brightness values. With the novel fluorescent probes, specific sensing...

  3. Single-stranded nucleic acid binding in Arabidopsis thaliana cold shock protein is cold shock domain dependent.

    Science.gov (United States)

    Mani, Ashutosh; Gupta, Dwijendra K

    2015-01-01

    Cold shock proteins (CSPs) are ancient nucleic acid-binding proteins and well conserved from bacteria to animals as well as plants. In prokaryotes, CSPs possess a single cold shock domain (CSD) while animal CSPs, flanked by N- and C-terminal domains, are commonly named Y-box proteins. Interestingly, the plants CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. The CSPs have been shown to play important role in development and stress adaptation in various plant species. The objective of this study was to find out the possible nucleic acid-binding affinities of whole CSP as well as independent domains, so that role of each individual domain may be revealed in Arabidopsis thaliana, the model plant species. The structure of CSP 3 protein from A. thaliana was modeled by homology-based approach and docking was done with different nucleic acid types.

  4. Medical Devices; Immunology and Microbiology Devices; Classification of the Streptococcus SPP. Nucleic Acid-Based Assay. Final order.

    Science.gov (United States)

    2017-10-30

    The Food and Drug Administration (FDA or we) is classifying the Streptococcus spp. nucleic acid-based assay into class II (special controls). The special controls that apply to the device type are identified in this order and will be part of the codified language for the Streptococcus spp. nucleic acid-based assay's classification. We are taking this action because we have determined that classifying the device into class II (special controls) will provide a reasonable assurance of safety and effectiveness of the device. We believe this action will also enhance patients' access to beneficial innovative devices, in part by reducing regulatory burdens.

  5. TD-DFT Investigation of the Magnetic Circular Dichroism Spectra of Some Purine and Pyrimidine Bases of Nucleic Acids

    DEFF Research Database (Denmark)

    Fahleson, Tobias; Kauczor, Joanna; Norman, Patrick

    2015-01-01

    We present a computational study of the magnetic circular dichroism (MCD) spectra in the 200–300 nm wavelength region of purine and its derivative hypoxanthine, as well as of the pyrimidine bases of nucleic acids uracil, thymine, and cytosine, using the B3LYP and CAM–B3LYP functionals. Solvent...... and the B term shape of the spectra of pyrimidine bases are reproduced. Our calculations also correctly reproduce the reversed phase of the MCD bands in purine compared to that of its derivatives present in nucleic acids. Solvent effects are sizable and system specific, but they do not in general alter...

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K(+) rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes......-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general....

  7. Immunotoxicity of nucleic acid reduced BioProtein - a bacterial derived single cell protein - in Wistar rats

    DEFF Research Database (Denmark)

    Mølck, Anne-marie; Poulsen, Morten; Christensen, Hanne Risager

    2002-01-01

    BioProtein is a single cell protein produced by a mixed methanotrophic and heterotrophic bacteria culture using natural gas as energy source, which has been approved for animal feed. BioProtein contains a large amount of nucleic acids making the product less suitable for human consumption......, therefore, a nucleic acid reduced variant (NABP) has been developed by the manufacturer. The purpose of the present study was to establish the safety of NABP in a subchronic toxicity rat study. Groups of 10 male and 10 female Wistar rats were fed diets containing 0, 6, 12 or 24% NABP for 13 weeks. Feeding...

  8. Insights into structure, dynamics and hydration of locked nucleic acid (LNA) strand-based duplexes from molecular dynamics simulations

    OpenAIRE

    Pande, Vineet; Nilsson, Lennart

    2008-01-01

    Locked nucleic acid (LNA) is a chemically modified nucleic acid with its sugar ring locked in an RNA-like (C3′-endo) conformation. LNAs show extraordinary thermal stabilities when hybridized with DNA, RNA or LNA itself. We performed molecular dynamics simulations on five isosequential duplexes (LNA–DNA, LNA–LNA, LNA–RNA, RNA–DNA and RNA–RNA) in order to characterize their structure, dynamics and hydration. Structurally, the LNA–DNA and LNA–RNA duplexes are found to be similar to regular RNA–D...

  9. Nucleic acid-based assays for the detection of high-risk human papillomavirus: a technical review.

    Science.gov (United States)

    Gibson, Jane S

    2014-09-01

    Nucleic acid-based high-risk human papillomavirus (hrHPV) testing is essential to contemporary cervical cancer screening. The numbers of commercially available assays approved by the US Food and Drug Administration for HPV nucleic acid detection have increased, each offering various approaches to analysis. An understanding of the methodologies associated with HPV testing is important to the practice of laboratory medicine. An overview of instruments, chemistries, laboratory workflows, and test limitations associated with current US Food and Drug Administration-approved assays is provided. © 2014 American Cancer Society.

  10. Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies.

    Science.gov (United States)

    Park, Jae-Seung; Surendran, Sneha; Kamendulis, Lisa M; Morral, Núria

    2011-01-18

    Primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as non-alcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. There is a need to identify simple methods to genetically manipulate primary hepatocytes and conduct functional studies with plasmids, small interfering RNA (siRNA) or microRNA (miRNA). New lipofection reagents are available that have the potential to yield higher levels of transfection with reduced toxicity. We have tested several liposome-based transfection reagents used in molecular biology research. We show that transfection efficiency with one of the most recently developed formulations, Metafectene Pro, is high with plasmid DNA (>45% cells) as well as double stranded RNA (>90% with siRNA or microRNA). In addition, negligible cytotoxicity was present with all of these nucleic acids, even if cells were incubated with the DNA:lipid complex for 16 hours. To provide the proof of concept that these conditions can be used not only for overexpression of a gene of interest, but also in RNA interference applications, we targeted two liver expressed genes, Sterol Regulatory Element-Binding Protein-1 and Fatty Acid Binding Protein 5 using plasmid-mediated short hairpin RNA expression. In addition, similar transfection conditions were used to optimally deliver siRNA and microRNA. We have identified a lipid-based reagent for primary hepatocyte transfection of nucleic acids currently used in molecular biology laboratories. The conditions described here can be used to expedite a large variety of research applications, from gene function studies to microRNA target identification.

  11. Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies

    Directory of Open Access Journals (Sweden)

    Morral Núria

    2011-01-01

    Full Text Available Abstract Background Primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as non-alcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. There is a need to identify simple methods to genetically manipulate primary hepatocytes and conduct functional studies with plasmids, small interfering RNA (siRNA or microRNA (miRNA. New lipofection reagents are available that have the potential to yield higher levels of transfection with reduced toxicity. Findings We have tested several liposome-based transfection reagents used in molecular biology research. We show that transfection efficiency with one of the most recently developed formulations, Metafectene Pro, is high with plasmid DNA (>45% cells as well as double stranded RNA (>90% with siRNA or microRNA. In addition, negligible cytotoxicity was present with all of these nucleic acids, even if cells were incubated with the DNA:lipid complex for 16 hours. To provide the proof of concept that these conditions can be used not only for overexpression of a gene of interest, but also in RNA interference applications, we targeted two liver expressed genes, Sterol Regulatory Element-Binding Protein-1 and Fatty Acid Binding Protein 5 using plasmid-mediated short hairpin RNA expression. In addition, similar transfection conditions were used to optimally deliver siRNA and microRNA. Conclusions We have identified a lipid-based reagent for primary hepatocyte transfection of nucleic acids currently used in molecular biology laboratories. The conditions described here can be used to expedite a large variety of research applications, from gene function studies to microRNA target identification.

  12. Nucleic Acid-Passivated Semiconductor Nanocrystals: Biomolecular Templating of Form and Function

    Science.gov (United States)

    Ma, Nan; Tikhomirov, Grigory; Kelley, Shana O.

    2009-01-01

    Conspectus Bright, photostable luminescent labels are powerful tools for the imaging of biological events in vitro and in vivo. Semiconductor nanocrystals have emerged as attractive alternatives to commonly used organic lumophores due to their high quantum yields and the spectral tunability that can be achieved through synthetic control. While conventional synthetic methods generally yield high-quality nanocrystals with excellent properties for biological imaging, ligand exchange and biological conjugation are necessary to make nanocrystals biocompatible and biospecific. These steps can result in substantial deterioration of optical characteristic of these nanocrystals. Moreover, the complexity of multistep nanocrystal synthesis, typically requiring inert and anhydrous conditions, prohibits many end users of these lumiphores from generating their own custom materials. We sought to streamline semiconductor nanocrystal synthesis and develop synthetic routes that would be accessible to scientists from all disciplines. In search of such an approach we turned to nucleic acids as a programmable and versatile ligand set, and found that these biomolecules are indeed appropriate for biocompatible semiconductor nanocrystals preparation. In this account we present a summary of our work on nucleic acids-programmed nanocrystal synthesis that has resulted in the successful development of a one-step synthesis of biofunctionalized nanocrystals in aqueous solution. We first discuss results obtained with nucleotide-capped cadmium and lead chalcogenide-based nanocrystals that served to guide further investigation of polynucleotide-assisted synthesis. We investigate the roles of individual nucleobases and their structures in passivation of the surfaces of nanocrystals and modulating morphology and optical characteristics. We show that nanocrystals’ optical properties and morphologies are highly influenced by nucleic acid structures and sequences, as well as by reaction conditions

  13. Unraveling Prion Protein Interactions with Aptamers and Other PrP-Binding Nucleic Acids.

    Science.gov (United States)

    Macedo, Bruno; Cordeiro, Yraima

    2017-05-17

    Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative disorders that affect humans and other mammals. The etiologic agents common to these diseases are misfolded conformations of the prion protein (PrP). The molecular mechanisms that trigger the structural conversion of the normal cellular PrP (PrP(C)) into the pathogenic conformer (PrP(Sc)) are still poorly understood. It is proposed that a molecular cofactor would act as a catalyst, lowering the activation energy of the conversion process, therefore favoring the transition of PrP(C) to PrP(Sc). Several in vitro studies have described physical interactions between PrP and different classes of molecules, which might play a role in either PrP physiology or pathology. Among these molecules, nucleic acids (NAs) are highlighted as potential PrP molecular partners. In this context, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) methodology has proven extremely valuable to investigate PrP-NA interactions, due to its ability to select small nucleic acids, also termed aptamers, that bind PrP with high affinity and specificity. Aptamers are single-stranded DNA or RNA oligonucleotides that can be folded into a wide range of structures (from harpins to G-quadruplexes). They are selected from a nucleic acid pool containing a large number (10(14)-10(16)) of random sequences of the same size (~20-100 bases). Aptamers stand out because of their potential ability to bind with different affinities to distinct conformations of the same protein target. Therefore, the identification of high-affinity and selective PrP ligands may aid the development of new therapies and diagnostic tools for TSEs. This review will focus on the selection of aptamers targeted against either full-length or truncated forms of PrP, discussing the implications that result from interactions of PrP with NAs, and their potential advances in the studies of prions. We will also provide a critical evaluation

  14. Emerging Utility of Urinary Cell-free Nucleic Acid Biomarkers for Prostate, Bladder, and Renal Cancers.

    Science.gov (United States)

    Lin, Selena Y; Linehan, Jennifer A; Wilson, Timothy G; Hoon, Dave S B

    2017-04-01

    By 2020 the estimated incidence of genitourinary (GU) cancers (prostate, bladder, and kidney) will be over 2 million worldwide and responsible for ∼800 000 deaths. Current diagnosis and monitoring methods of GU cancer patients are often invasive and/or lack sensitivity and specificity. Given the utility of blood-based cell-free nucleic acid (cfNA) biomarkers, the development of urinary cfNA biomarkers may improve the sensitivity of urine assays utilizing urine sediment for GU cancers. This review of urinary cfNA in GU cancers identifies the current stage of research, potential clinical utility, and the next steps needed to enter clinical use. To critically evaluate the literature of urinary cfNA in GU cancers for clinical utility in diagnosis, screening, and precision medicine. Furthermore, the strategy for future efforts to discover potential new urinary cfNA biomarkers will be described. A PubMed database (2006 to current) search was performed according to Preferred Reporting Items for Systemic Review and Meta-analysis using key Medical Subject Headings terms. Additional studies were obtained by cross-referencing from the literature. The collective research publications in urinary cfNA of GU cancers present a promising alternative liquid biopsy approach compared with blood biopsies and urine sediment, particularly for early-stage GU diseases. Urinary cfNA as a liquid biopsy holds potential for a more sensitive alternative to blood biopsies and urine sediment-based tests for clinical use in GU cancers. Not only does urinary cfNA offer advantages including the potential for more frequent testing, monitoring, and home use, but also has applications in early-stage GU cancers. In this review, we evaluated the current status of urinary cell-free nucleic acid in genitourinary cancers. We identified the potential advantages of urinary cell-free nucleic acid over blood and urine sediment and its clinical use in genitourinary cancer. Copyright © 2017 European

  15. Cationic, amphiphilic copolymer micelles as nucleic acid carriers for enhanced transfection in rat spinal cord.

    Science.gov (United States)

    Gwak, So-Jung; Nice, Justin; Zhang, Jeremy; Green, Benjamin; Macks, Christian; Bae, Sooneon; Webb, Ken; Lee, Jeoung Soo

    2016-04-15

    Spinal cord injury commonly leads to permanent motor and sensory deficits due to the limited regenerative capacity of the adult central nervous system (CNS). Nucleic acid-based therapy is a promising strategy to deliver bioactive molecules capable of promoting axonal regeneration. Branched polyethylenimine (bPEI: 25kDa) is one of the most widely studied nonviral vectors, but its clinical application has been limited due to its cytotoxicity and low transfection efficiency in the presence of serum proteins. In this study, we synthesized cationic amphiphilic copolymers, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP), by grafting low molecular weight PLGA (4kDa) to bPEI (25kDa) at approximately a 3:1 ratio as an efficient nonviral vector. We show that PgP micelle is capable of efficiently transfecting plasmid DNA (pDNA) and siRNA in the presence of 10% serum in neuroglioma (C6) cells, neuroblastoma (B35) cells, and primary E8 chick forebrain neurons (CFN) with pDNA transfection efficiencies of 58.8%, 75.1%, and 8.1%, respectively. We also show that PgP provides high-level transgene expression in the rat spinal cord in vivo that is substantially greater than that attained with bPEI. The combination of improved transfection and reduced cytotoxicity in vitro in the presence of serum and in vivo transfection of neural cells relative to conventional bPEI suggests that PgP may be a promising nonviral vector for therapeutic nucleic acid delivery for neural regeneration. Gene therapy is a promising strategy to overcome barriers to axonal regeneration in the injured central nervous system. Branched polyethylenimine (bPEI: 25kDa) is one of the most widely studied nonviral vectors, but its clinical application has been limited due to cytotoxicity and low transfection efficiency in the presence of serum proteins. Here, we report cationic amphiphilic copolymers, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) that are capable of efficiently transfecting reporter

  16. Adsorption of nucleic acid bases and amino acids on single-walled carbon and boron nitride nanotubes: a first-principles study.

    Science.gov (United States)

    Zheng, Jiaxin; Song, Wei; Wang, Lu; Lu, Jing; Luo, Guangfu; Zhou, Jing; Qin, Rui; Li, Hong; Gao, Zhengxiang; Lai, Lin; Li, Guangping; Mei, Wai Ning

    2009-11-01

    We study the adsorptions of nucleic acid bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) and four amino acids phenylalanine, tyrosine, tryptophan, alanine on the single-walled carbon nanotubes (SWCNTs) and boron nitride nanotubes (SWBNNTs) by using density functional theory. We find that the aromatic content plays a critical role in the adsorption. The adsorptions of nucleic acid bases and amino acids on the (7, 7) SWBNNT are stronger than those on the (7, 7) SWCNT. Oxidative treatment of SWCNTs favors the adsorption of biomolecules on nanotubes.

  17. Sensitivity of individual and mini-pool nucleic acid testing assessed by dilution of hepatitis B nucleic acid testing yield samples

    Directory of Open Access Journals (Sweden)

    Kabita Chatterjee

    2014-01-01

    Full Text Available Introduction: For nucleic acid testing (NAT of blood donations, either the blood samples can be pooled together in a batch of six or eight prior to testing (mini-pool-NAT [MP-NAT], or the tests can be run on every individual sample (individual donor-NAT [ID-NAT]. It has been debated in various studies whether pooling of samples results in decreased sensitivity of detection as the volume of individual samples gets lesser in a pool. The objective of this study was to investigate the effect of dilution on the sensitivity of tests. Materials and Methods: The study was performed on nine plasma samples which were hepatitis B reactive exclusively by Procleix Ultrio Plus and not by Procleix Ultrio or serology. These nine exclusive UltrioPlus ID-NAT yield samples were diluted in 1:2, 1:4. 1:6 and 1:8 dilutions using previously tested negative plasma and each dilution of every sample along with archived undiluted sample were retested in three replicates with Procleix Ultrio Plus Assay. Results: Among NAT yield samples, 88.88% of the samples were detected when retested in ID-NAT in undiluted form. Samples with higher viral load (sample 5 and 6 were detected by all dilutions. When samples with viral load below 20 IU/mL were tested in dilutions of 1:6 or 1:8, only 9 out of 27 replicates (33.33% were detected. This means that more than 67% of low viral load samples were missed by MP-NAT of 1:6 or 1:8 dilution out of total NAT yield samples. Conclusion: Individual Donor NAT is ideal methodology for NAT as dilution due to pooling may miss samples with low viral load as evident in this study.

  18. Sensitivity of individual and mini-pool nucleic acid testing assessed by dilution of hepatitis B nucleic acid testing yield samples.

    Science.gov (United States)

    Chatterjee, Kabita; Agarwal, Nitin; Coshic, Poonam; Borgohain, Mayuri; Chakroborty, Sourit

    2014-01-01

    For nucleic acid testing (NAT) of blood donations, either the blood samples can be pooled together in a batch of six or eight prior to testing (mini-pool-NAT [MP-NAT]), or the tests can be run on every individual sample (individual donor-NAT [ID-NAT]). It has been debated in various studies whether pooling of samples results in decreased sensitivity of detection as the volume of individual samples gets lesser in a pool. The objective of this study was to investigate the effect of dilution on the sensitivity of tests. The study was performesd on nine plasma samples which were hepatitis B reactive exclusively by Procleix Ultrio Plus and not by Procleix Ultrio or serology. These nine exclusive UltrioPlus ID-NAT yield samples were diluted in 1:2, 1:4. 1:6 and 1:8 dilutions using previously tested negative plasma and each dilution of every sample along with archived undiluted sample were retested in three replicates with Procleix Ultrio Plus Assay. Among NAT yield samples, 88.88% of the samples were detected when retested in ID-NAT in undiluted form. Samples with higher viral load (sample 5 and 6) were detected by all dilutions. When samples with viral load below 20 IU/mL were tested in dilutions of 1:6 or 1:8, only 9 out of 27 replicates (33.33%) were detected. This means that more than 67% of low viral load samples were missed by MP-NAT of 1:6 or 1:8 dilution out of total NAT yield samples. Individual Donor NAT is ideal methodology for NAT as dilution due to pooling may miss samples with low viral load as evident in this study.

  19. X-ray crystallographic visualization of drug-nucleic acid intercalative binding: structure of an ethidium-dinucleoside monophosphate crystalline complex, ethidium: 5-iodouridylyl(3'-5')adenosine (drug-nucleic acid interactions/intercalation/double helix unwinding)

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, C.C.; Jain, S.C.; Sobell, H.M.

    1975-02-01

    The drug ethidium bromide has been cocrystallized with the dinucleoside monophosphate 5-iodouridylyl (3'-5')adenosine. The three-dimensional structure to atomic resolution by x-ray crystallography was solved. This has allowed the direct visualization of intercalative binding by this drug to a fragment of a nucleic acid double helix.

  20. Nucleic-acid characterization of the identity and activity of subsurface microorganisms

    Science.gov (United States)

    Madsen, E. L.

    Nucleic-acid approaches to characterizing naturally occurring microorganisms in their habitats have risen to prominence during the last decade. Extraction of deoxyribonucleic-acid (DNA) and ribonucleic-acid (RNA) biomarkers directly from environmental samples provides a new means of gathering information in microbial ecology. This review article defines: (1) the subsurface habitat; (2) what nucleic-acid procedures are; and (3) the types of information nucleic-acid procedures can and cannot reveal. Recent literature examining microbial nucleic acids in the terrestrial subsurface is tabulated and reviewed. The majority of effort to date has focused upon insights into the identity and phylogeny of subsurface microorganisms afforded by analysis of their 16S rRNA genes. Given the power of nucleic-acid-based procedures and their limited application to subsurface habitats to date, many future opportunities await exploration. Au cours des derniers dix ans, les approches basées sur les acides nucléiques sont apparues et devenues essentielles pour caractériser dans leurs habitats les microorganismes existant à l'état naturel. L'extraction directe de l'ADN et de l'ARN, qui sont des biomarqueurs, d'échantillons environnementaux a fourni un nouveau moyen d'obtenir des informations sur l'écologie microbienne. Cet article synthétique définit 1) l'habitat souterrain, 2) ce que sont les procédures basées sur les acides nucléiques, 3) quel type d'informations ces procéedures peuvent et ne peuvent pas révéler. Les travaux récemment publiés concernatn les acides nucléiques microbiens dans le milieu souterrain terrestre sont catalogués et passés en revue. La majorité des efforts pour obtenir es données s'est concentrée sur l'identité et la phylogénie des microorganismes souterrains fournies par l'analyse de leurs gènes 16S rRNA. Étant donné la puissance des procédures basées sur les acides nucléiques et leur application limitée aux habitats souterrains

  1. Immunologic, spectrophotometric and nucleic acid based methods for the detection and quantification of airborne pollen.

    Science.gov (United States)

    Rittenour, William R; Hamilton, Robert G; Beezhold, Donald H; Green, Brett J

    2012-09-28

    Microscopic identification of pollen morphological phenotypes has been the traditional method used to identify and quantify pollen collected by air monitoring stations worldwide. Although this method has enabled a semi-standardized approach for the assessment of pollen exposure, limitations including labor intensiveness, required expertise, examiner bias, and the inability to differentiate species, genera, and in some cases families have limited data derived from the these stations. Recent advances in chemical, biochemical and molecular detection methods have provided standardized alternatives to this microscopic approach. In this review, we examine the applicability of alternative methodologies, in particular nucleic acid based assays involving the quantitative polymerase chain reaction, for the standardized detection of airborne pollen. Published by Elsevier B.V.

  2. Thermal Stability of Modified i-Motif Oligonucleotides with Naphthalimide Intercalating Nucleic Acids

    DEFF Research Database (Denmark)

    El-Sayed, Ahmed Ali; Pedersen, Erik B.; Khaireldin, Nahid Y.

    2016-01-01

    of naphthalimide (1H-benzo[de]isoquinoline-1,3(2H)-dione) as the intercalating nucleic acid. The stabilities of i-motif structures with inserted naphthalimide intercalating nucleotides were studied using UV melting temperatures (Tm) and circular dichroism spectra at different pH values and conditions (crowding......In continuation of our investigation of characteristics and thermodynamic properties of the i-motif 5′-d[(CCCTAA)3CCCT)] upon insertion of intercalating nucleotides into the cytosine-rich oligonucleotide, this article evaluates the stabilities of i-motif oligonucleotides upon insertion...... and non-crowding). This study indicated a positive effect of the naphthalimide intercalating nucleotides on the stabilities of the i-motif structures compared to the wild-type structure which is in contrast to a previous observation for a pyrene-intercalating nucleotide showing a decrease in Tm values....

  3. Single-Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking.

    Science.gov (United States)

    Kawai, Kiyohiko; Miyata, Takafumi; Shimada, Naohiko; Ito, Syoji; Miyasaka, Hiroshi; Maruyama, Atsushi

    2017-11-27

    Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to investigate the dynamics of biomolecular events in real time. However, it requires two fluorophores and can be applied only to dynamics that accompany large changes in distance between the molecules. Herein, we introduce a method for kinetic analysis based on control of fluorescence blinking (KACB), a general approach to investigate the dynamics of biomolecules by using a single fluorophore. By controlling the kinetics of the redox reaction the blinking kinetics or pattern can be controlled to be affected by microenvironmental changes around a fluorophore (rKACB), thereby enabling real-time single-molecule measurement of the structure-changing dynamics of nucleic acids. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  4. Single‐Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking

    Science.gov (United States)

    Miyata, Takafumi; Shimada, Naohiko; Ito, Syoji; Miyasaka, Hiroshi

    2017-01-01

    Abstract Single‐molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to investigate the dynamics of biomolecular events in real time. However, it requires two fluorophores and can be applied only to dynamics that accompany large changes in distance between the molecules. Herein, we introduce a method for kinetic analysis based on control of fluorescence blinking (KACB), a general approach to investigate the dynamics of biomolecules by using a single fluorophore. By controlling the kinetics of the redox reaction the blinking kinetics or pattern can be controlled to be affected by microenvironmental changes around a fluorophore (rKACB), thereby enabling real‐time single‐molecule measurement of the structure‐changing dynamics of nucleic acids. PMID:28990725

  5. Ultramild protein-mediated click chemistry creates efficient oligonucleotide probes for targeting and detecting nucleic acids

    DEFF Research Database (Denmark)

    Nåbo, Lina J.; Madsen, Charlotte Stahl; Jensen, Knud Jørgen

    2015-01-01

    Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid...... conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine-based fluorescent probes in vitro and rationalize our...... results by electronic structure calculations. Functionalized oligonucleotides were prepared in good yields by protein-mediated CuAAC click reactions for the first time with a human copper-binding chaperon. The carbohydrate, peptide, and fluorescent derivatives display high binding affinity and selectivity...

  6. Surface plasmon field-enhanced fluorescence spectroscopy in PCR product analysis by peptide nucleic acid probes.

    Science.gov (United States)

    Yao, Danfeng; Yu, Fang; Kim, Junyoung; Scholz, Judith; Nielsen, Peter E; Sinner, Eva-Kathrin; Knoll, Wolfgang

    2004-12-14

    Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) was recently developed for PCR product analysis, which allowed for real-time monitoring of hybridization processes and for the detection of trace amounts of PCR products, with a detection limit of 100 fmol on the peptide nucleic acid (PNA) probe surface, and 500 fmol on the DNA probe surface. By selectively labeling the strands of PCR-amplified DNA, it was shown that the heat denaturation process in combination with the application of low-salt condition substantially reduced the interference from the antisense strands and thus simplified the surface hybridization. Furthermore, SPFS was demonstrated to be capable of quantitatively discriminating the difference induced by single nucleotide substitution, even within one minute of contact time.

  7. Aptamer- and nucleic acid enzyme-based systems for simultaneous detection of multiple analytes

    Science.gov (United States)

    Lu, Yi [Champaign, IL; Liu, Juewen [Albuquerque, NM

    2011-11-15

    The present invention provides aptamer- and nucleic acid enzyme-based systems for simultaneously determining the presence and optionally the concentration of multiple analytes in a sample. Methods of utilizing the system and kits that include the sensor components are also provided. The system includes a first reactive polynucleotide that reacts to a first analyte; a second reactive polynucleotide that reacts to a second analyte; a third polynucleotide; a fourth polynucleotide; a first particle, coupled to the third polynucleotide; a second particle, coupled to the fourth polynucleotide; and at least one quencher, for quenching emissions of the first and second quantum dots, coupled to the first and second reactive polynucleotides. The first particle includes a quantum dot having a first emission wavelength. The second particle includes a second quantum dot having a second emission wavelength different from the first emission wavelength. The third polynucleotide and the fourth polynucleotide are different.

  8. Modeling the interaction between dendrimers and nucleic acids: a molecular perspective through hierarchical scales.

    Science.gov (United States)

    Pavan, Giovanni M

    2014-12-01

    Cationic dendrimers are promising nanocarriers for gene delivery thanks to their ability to establish strong interactions with oppositely charged strands of DNA and siRNA and to promote their aggregation. The binding between dendrimers and nucleic acids is typically a complex process that involves various types of interactions at different scales. To design efficient dendrimer candidates for DNA and siRNA binding it is necessary to have a detailed understanding of their interactions with oligonucleotides in the solvent. Molecular simulation can support experimental work, providing a privileged point of view on the aggregation process. This Minireview discusses recent computational efforts to unravel dendrimer-oligonucleotide binding, and proposes a perspective of the multiscale aggregation process based on hierarchy and on the transformations of the interacting "molecular units" following intermolecular interactions. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Importance of databases of nucleic acids for bioinformatic analysis focused to genomics

    Science.gov (United States)

    Jimenez-Gutierrez, L. R.; Barrios-Hernández, C. J.; Pedraza-Ferreira, G. R.; Vera-Cala, L.; Martinez-Perez, F.

    2016-08-01

    Recently, bioinformatics has become a new field of science, indispensable in the analysis of millions of nucleic acids sequences, which are currently deposited in international databases (public or private); these databases contain information of genes, RNA, ORF, proteins, intergenic regions, including entire genomes from some species. The analysis of this information requires computer programs; which were renewed in the use of new mathematical methods, and the introduction of the use of artificial intelligence. In addition to the constant creation of supercomputing units trained to withstand the heavy workload of sequence analysis. However, it is still necessary the innovation on platforms that allow genomic analyses, faster and more effectively, with a technological understanding of all biological processes.

  10. Well-defined Cationic Shell Crosslinked Nanoparticles for Efficient Delivery of DNA or Peptide Nucleic Acids

    Science.gov (United States)

    Zhang, Ke; Fang, Huafeng; Shen, Gang; Taylor, John-Stephen A.; Wooley, Karen L.

    2009-01-01

    This mini-review highlights developments that have been made over the past year to advance the construction of well-defined nanoscale objects to serve as devices for cell transfection. Design of the nanoscale objects originated from biomimicry concepts, using histones as the model, to afford cationic shell crosslinked knedel-like (cSCK) nanoparticles. Packaging and delivery of plasmid DNA, oligonucleotides, and peptide nucleic acids were studied by dynamic light scattering, transmission electron microscopy, gel electrophoresis, biological activity assays, RT-PCR measurements, flow cytometry, and confocal fluorescence microscopy. With the demonstration of more efficient cell transfection in vitro than that achieved using commercially-available transfection agents, together with the other features offered by the robust nanostructural framework, work continues toward the application of these cSCKs for in vivo molecular recognition of genetic material, for imaging and therapy targeted specifically to pulmonary injury and disease. PMID:19687218

  11. TDDFT Study of the Optical Excitation of Nucleic Acid Bases-C60 Complexes.

    Science.gov (United States)

    Tawfik, Sherif Abdulkader; Cui, X Y; Ringer, S P; Stampfl, C

    2017-11-30

    The potential of C60 as a nucleic acid base (NAB) optical sensor is theoretically explored. We investigate the adsorption of four NABs, namely, adenine, cytosine, guanine, and thymine, on C60 in the gas phase. For the optimal NAB@C60 adsorption configurations, obtained using a dispersion-corrected density functional, we calculate the vis-near-ultraviolet optical response using time-dependent density functional theory. While the isolated C60 and NAB molecules do not exhibit visible optical excitation, we find that C60/NAB conjugation gives rise to distinct spectral features in the visible range. These results suggest that C60 conjugation can be applied for photodetection of individual NABs.

  12. Visualizing nucleic acids in living cells by fluorescence in vivo hybridization.

    Science.gov (United States)

    Wiegant, Joop; Brouwer, Anneke K; Tanke, Hans J; Dirks, Roeland W

    2010-01-01

    The analysis of the spatial-dynamic properties of DNA and RNA molecules in living cells will greatly extend our knowledge of genome organization and gene expression regulation in the cell nucleus. The development of hybridization methods allowing detection of specific endogenous DNA and RNA sequences in living cells has therefore been a challenge for many years. However, there are many technical issues that have proven so far to be difficult, or even impossible, to overcome. As a result, in most situations, the application of in vivo hybridization methods is currently limited to the visualization of highly repetitive DNA sequences or abundant RNA species. We describe a protocol that enables the visualization and tracking of telomeres in living cells by hybridization with a fluorescent peptide nucleic acid (PNA) probe. Furthermore, we describe a method that allows the detection of abundant endogenous RNAs in living cells by microinjecting fluorescently labeled complementary 2'-O-methyl RNA probes.

  13. Circulating nucleic acids in plasma and serum: applications in diagnostic techniques for noninvasive prenatal diagnosis

    Directory of Open Access Journals (Sweden)

    Gahan PB

    2013-04-01

    Full Text Available Peter B Gahan Anatomy and Human Sciences Department, King's College London, London Bridge, London, UK Abstract: The analysis of fetal nucleic acids in maternal blood 13 years ago has led to the initiation of noninvasive methods for the early determination of fetal gender, rhesus D status, and a number of aneuploid disorders and hemoglobinopathies. Subsequently, a comparatively large quantity of fetal DNA and RNA has been demonstrated in amniotic fluid as well as small amounts in premature infant saliva. The DNA and RNA in amniotic fluid has permitted an analysis of core transcriptomes, whilst the DNA and RNA in saliva allows the early detection and treatment monitoring of fetal developmental problems. These aspects are discussed together with the methodology and limits of analysis for noninvasive prenatal diagnosis in predictive, preventive, and personalized medicine. Keywords: fetal circulating DNA/RNA, amniotic fluid, saliva, aneuploidy, thalassemias

  14. Biomedical Applications of Quantum Dots, Nucleic Acid-Based Aptamers, and Nanostructures in Biosensors.

    Science.gov (United States)

    Meshik, Xenia; Farid, Sidra; Choi, Min; Lan, Yi; Mukherjee, Souvik; Datta, Debopam; Dutta, Mitra; Stroscio, Michael A

    2015-01-01

    This review is a survey of the biomedical applications of semiconductor quantum dots, nucleic acid-based aptamers, and nanosensors as molecular biosensors. It focuses on the detection of analytes in biomedical applications using (1) advances in molecular beacons incorporating semiconductor quantum dots and nanoscale quenching elements; (2) aptamer-based nanosensors on a variety of platforms, including graphene; (3) Raman scattering and surface-enhanced Raman scattering (SERS) using nanostructures for enhanced SERS spectra of biomolecules, including aptamers; and (4) the electrical and optical properties of nanostructures incorporated into molecular beacons and aptamer-based nanosensors. Research done at the University of Illinois at Chicago (UIC) is highlighted throughout since it emphasizes the specific approaches taken by the bioengineering department at UIC.

  15. The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers.

    Directory of Open Access Journals (Sweden)

    Nancy A Stearns

    Full Text Available Antibodies to DNA (anti-DNA are the serological hallmark of systemic lupus erythematosus (SLE and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3, hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

  16. The free energy of locking a ring: Changing a deoxyribonucleoside to a locked nucleic acid.

    Science.gov (United States)

    Xu, You; Villa, Alessandra; Nilsson, Lennart

    2017-06-05

    Locked nucleic acid (LNA), a modified nucleoside which contains a bridging group across the ribose ring, improves the stability of DNA/RNA duplexes significantly, and therefore is of interest in biotechnology and gene therapy applications. In this study, we investigate the free energy change between LNA and DNA nucleosides. The transformation requires the breaking of the bridging group across the ribose ring, a problematic transformation in free energy calculations. To address this, we have developed a 3-step (easy to implement) and a 1-step protocol (more efficient, but more complicated to setup), for single and dual topologies in classical molecular dynamics simulations, using the Bennett Acceptance Ratio method to calculate the free energy. We validate the approach on the solvation free energy difference for the nucleosides thymidine, cytosine, and 5-methyl-cytosine. © 2017 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. © 2017 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

  17. Anionic magnetite nanoparticle conjugated with pyrrolidinyl peptide nucleic acid for DNA base discrimination

    Energy Technology Data Exchange (ETDEWEB)

    Khadsai, Sudarat; Rutnakornpituk, Boonjira [Naresuan University, Department of Chemistry and Center of Excellence in Biomaterials, Faculty of Science (Thailand); Vilaivan, Tirayut [Chulalongkorn University, Department of Chemistry, Organic Synthesis Research Unit, Faculty of Science (Thailand); Nakkuntod, Maliwan [Naresuan University, Department of Biology, Faculty of Science (Thailand); Rutnakornpituk, Metha, E-mail: methar@nu.ac.th [Naresuan University, Department of Chemistry and Center of Excellence in Biomaterials, Faculty of Science (Thailand)

    2016-09-15

    Magnetite nanoparticles (MNPs) were surface modified with anionic poly(N-acryloyl glycine) (PNAG) and streptavidin for specific interaction with biotin-conjugated pyrrolidinyl peptide nucleic acid (PNA). Hydrodynamic size (D{sub h}) of PNAG-grafted MNPs varied from 334 to 496 nm depending on the loading ratio of the MNP to NAG in the reaction. UV–visible and fluorescence spectrophotometries were used to confirm the successful immobilization of streptavidin and PNA on the MNPs. About 291 pmol of the PNA/mg MNP was immobilized on the particle surface. The PNA-functionalized MNPs were effectively used as solid supports to differentiate between fully complementary and non-complementary/single-base mismatch DNA using the PNA probe. These novel anionic MNPs can be efficiently applicable for use as a magnetically guidable support for DNA base discrimination.Graphical Abstract.

  18. Urinary markers of nucleic acid oxidation and cancer in type 2 diabetes

    DEFF Research Database (Denmark)

    Broedbaek, Kasper; Siersma, Volkert; Henriksen, Trine Foged

    2015-01-01

    with breast cancer in the crude analyses (unadjusted hazard ratio for breast cancer per natural log increase in 8-oxodG was 2.37 [95% CI, 1.07-5.26]), although the association was attenuated in the adjusted analyses (sex- and age-adjusted hazard ratio 2.15 [95% CI, 0.92-5.02] and multivariate adjusted hazard......AIMS/HYPOTHESIS: We investigated whether urinary markers of nucleic acid oxidation are associated with an increased risk of cancer in type 2 diabetes patients. METHODS: Urine samples from 1381 newly diagnosed diabetes patients were assayed for the oxidatively modified guanine nucleosides 8-oxo-7......,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo). Cox proportional hazards regression was used to examine the relationship between the urinary markers and cancer incidence. RESULTS: The crude analyses showed an association between overall cancer and urinary excretion...

  19. Peptides, polypeptides and peptide-polymer hybrids as nucleic acid carriers.

    Science.gov (United States)

    Ahmed, Marya

    2017-10-24

    Cell penetrating peptides (CPPs), and protein transduction domains (PTDs) of viruses and other natural proteins serve as a template for the development of efficient peptide based gene delivery vectors. PTDs are sequences of acidic or basic amphipathic amino acids, with superior membrane trespassing efficacies. Gene delivery vectors derived from these natural, cationic and cationic amphipathic peptides, however, offer little flexibility in tailoring the physicochemical properties of single chain peptide based systems. Owing to significant advances in the field of peptide chemistry, synthetic mimics of natural peptides are often prepared and have been evaluated for their gene expression, as a function of amino acid functionalities, architecture and net cationic content of peptide chains. Moreover, chimeric single polypeptide chains are prepared by a combination of multiple small natural or synthetic peptides, which imparts distinct physiological properties to peptide based gene delivery therapeutics. In order to obtain multivalency and improve the gene delivery efficacies of low molecular weight cationic peptides, bioactive peptides are often incorporated into a polymeric architecture to obtain novel 'polymer-peptide hybrids' with improved gene delivery efficacies. Peptide modified polymers prepared by physical or chemical modifications exhibit enhanced endosomal escape, stimuli responsive degradation and targeting efficacies, as a function of physicochemical and biological activities of peptides attached onto a polymeric scaffold. The focus of this review is to provide comprehensive and step-wise progress in major natural and synthetic peptides, chimeric polypeptides, and peptide-polymer hybrids for nucleic acid delivery applications.

  20. Electrostatic binding and hydrophobic collapse of peptide-nucleic acid aggregates quantified using force spectroscopy.

    Science.gov (United States)

    Camunas-Soler, Joan; Frutos, Silvia; Bizarro, Cristiano V; de Lorenzo, Sara; Fuentes-Perez, Maria Eugenia; Ramsch, Roland; Vilchez, Susana; Solans, Conxita; Moreno-Herrero, Fernando; Albericio, Fernando; Eritja, Ramón; Giralt, Ernest; Dev, Sukhendu B; Ritort, Felix

    2013-06-25

    Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g., Alzheimer's and Parkinson's diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide that contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interactions. From the measured pulling curves we determine the spectrum of binding affinities, kinetic barriers, and lengths of DNA segments sequestered within the KF-DNA complex. We find there is a capture distance beyond which the complex collapses into compact aggregates stabilized by strong hydrophobic forces and discuss how the bending rigidity of the nucleic acid affects this process. We hypothesize that within an in vivo context, the enhanced electrostatic interaction of KF due to its aggregation might mediate the binding to other polyanions. The proposed methodology should be useful to quantitatively characterize other compounds or proteins in which the formation of aggregates is relevant.