WorldWideScience

Sample records for bridged nucleic acid

  1. Cleavage of nucleic acids

    Energy Technology Data Exchange (ETDEWEB)

    Prudent, James R. (Madison, WI); Hall, Jeff G. (Madison, WI); Lyamichev, Victor L. (Madison, WI); Brow, Mary Ann D. (Madison, WI); Dahlberg, James E. (Madison, WI)

    2007-12-11

    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.

  2. Cleavage of nucleic acids

    Energy Technology Data Exchange (ETDEWEB)

    Prudent, James R. (Madison, WI); Hall, Jeff G. (Waunakee, WI); Lyamichev, Victor I. (Madison, WI); Brow; Mary Ann D. (Madison, WI); Dahlberg, James E. (Madison, WI)

    2010-11-09

    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.

  3. Nucleic acid detection compositions

    Energy Technology Data Exchange (ETDEWEB)

    Prudent, James R. (Madison, WI); Hall, Jeff G. (Madison, WI); Lyamichev, Victor I. (Madison, WI); Brow, Mary Ann (Madison, WI); Dahlberg, James L. (Madison, WI)

    2008-08-05

    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.

  4. Nucleic acid detection assays

    Energy Technology Data Exchange (ETDEWEB)

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann; Dahlberg, James E.

    2005-04-05

    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.

  5. Cleavage of nucleic acids

    Science.gov (United States)

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    2000-01-01

    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.

  6. Nucleic acid detection kits

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann; Kwiatkowski, Robert W.; Vavra, Stephanie H.

    2005-03-29

    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 nucleic acid from various viruses in a sample.

  7. 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....

  8. 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...

  9. 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...

  10. 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....

  11. 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....

  12. 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....

  13. Method for isolating nucleic acids

    Science.gov (United States)

    Hurt, Jr., Richard Ashley; Elias, Dwayne A.

    2015-09-29

    The current disclosure provides methods and kits for isolating nucleic acid from an environmental sample. The current methods and compositions further provide methods for isolating nucleic acids by reducing adsorption of nucleic acids by charged ions and particles within an environmental sample. The methods of the current disclosure provide methods for isolating nucleic acids by releasing adsorbed nucleic acids from charged particles during the nucleic acid isolation process. The current disclosure facilitates the isolation of nucleic acids of sufficient quality and quantity to enable one of ordinary skill in the art to utilize or analyze the isolated nucleic acids for a wide variety of applications including, sequencing or species population analysis.

  14. Neutron Nucleic Acid Crystallography.

    Science.gov (United States)

    Chatake, Toshiyuki

    2016-01-01

    The hydration shells surrounding nucleic acids and hydrogen-bonding networks involving water molecules and nucleic acids are essential interactions for the structural stability and function of nucleic acids. Water molecules in the hydration shells influence various conformations of DNA and RNA by specific hydrogen-bonding networks, which often contribute to the chemical reactivity and molecular recognition of nucleic acids. However, X-ray crystallography could not provide a complete description of structural information with respect to hydrogen bonds. Indeed, X-ray crystallography is a powerful tool for determining the locations of water molecules, i.e., the location of the oxygen atom of H2O; however, it is very difficult to determine the orientation of the water molecules, i.e., the orientation of the two hydrogen atoms of H2O, because X-ray scattering from the hydrogen atom is very small.Neutron crystallography is a specialized tool for determining the positions of hydrogen atoms. Neutrons are not diffracted by electrons, but are diffracted by atomic nuclei; accordingly, neutron scattering lengths of hydrogen and its isotopes are comparable to those of non-hydrogen atoms. Therefore, neutron crystallography can determine both of the locations and orientations of water molecules. This chapter describes the current status of neutron nucleic acid crystallographic research as well as the basic principles of neutron diffraction experiments performed on nucleic acid crystals: materials, crystallization, diffraction experiments, and structure determination. PMID:26227050

  15. Invasive cleavage of nucleic acids

    Energy Technology Data Exchange (ETDEWEB)

    Prudent, James R. (Madison, WI); Hall, Jeff G. (Madison, WI); Lyamichev, Victor I. (Madison, WI); Brow, Mary Ann D. (Madison, WI); Dahlberg, James E. (Madison, WI)

    2002-01-01

    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.

  16. Invasive cleavage of nucleic acids

    Science.gov (United States)

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    1999-01-01

    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.

  17. Electrochemistry of nucleic acids

    Czech Academy of Sciences Publication Activity Database

    Paleček, Emil; Bartošík, Martin

    2012-01-01

    Roč. 112, č. 6 (2012), s. 3427-3481. ISSN 0009-2665 R&D Projects: GA ČR(CZ) GAP301/11/2055; GA MŠk(CZ) ME09038; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : nucleic acids electrochemistry * DNA biosensors * DNA damage Subject RIV: BO - Biophysics Impact factor: 41.298, year: 2012

  18. Nucleic Acid Vaccines

    Institute of Scientific and Technical Information of China (English)

    LU Shan

    2004-01-01

    @@ Anew method of immunization was discovered in the early 1990s. Several research groups independently demonstrated that direct inoculation of DNA plasmids coding for a specific protein antigen could elicit immune responses against that antigen[1-4].Since in theory the mRNA molecules also have the potential to be translated into the protein antigen, this vaccination approach was officially named by WHO as the nucleic acid vaccination even though the term DNA vaccine has been used more commonly in the literature. This novel approach is considered the fourth generation of vaccines after live attenuated vaccines, killed or inactivated vaccines and recombinant protein based subunit vaccines.

  19. Origin of nucleic acids

    International Nuclear Information System (INIS)

    The appearance of nucleic acids is the first event after the birth of membranes which made it possible to assure the perenniality of information. The complexity of these molecules has led some scientists to propose that they were not prebiotic but rather derived a more simple and achiral primitive ancestor. This hypothesis suggests that ribose possesses properties that allowed the formation of certain polysaccharides which evolved to RNA. The first step of the hypothesis is the selection and concentration of ribofuranose. This sugar has chelating properties and its alpha-ribofuranose is favoured in the chelating position. The density of the sugar with a heavy cation is greater than water and thus the complex can escape the UV radiation at the surface of the ocean. The particularity of ribose is to be able to form a homochiral regular array of these basic chelating structures with pyrophosphite. These arrays evolve towards the formation of polysaccharides (poly ribose phosphate) which have a very organized structure. These polysaccharides in turn evolve to RNA by binding of adenine and deoxyguanine which are HCN derivatives that can react with the polysaccharides. The primitive RNA is methylated and oxidized to form prebiotic RNA with adenosine, cytidine, 7methyl-guanosine and ribothymidine as nucleic bases. The pathway of biosynthesis of DNA form RNA will be studied. I suggest that the appearance of DNA results form the interaction between prebiotic double stranded RNA and proteins. DNA could be a product of RNA degradation by proteins. The catabolism of RNA to DNA requires a source of free radicals, protons and hydrides. RNA cannot produce free radicals, which are provided by the phenol group of the amino acid tyrosien. Protons are provided by the medium and hydrides are provided by 7-methyl-guanosine which can fix hydrides coming from hydrogen gas and donate them for the transformation of a riboside to a deoxyriboside. This pathway suggests that DNA appeared at

  20. The Nucleic Acid Database (NDB)

    Czech Academy of Sciences Publication Activity Database

    Berman, H. M.; Feng, Z.; Schneider, Bohdan; Westbrook, J.; Zardecki, C.

    Vol. F. Dordrecht : Kluwer Academic, 2001 - (Rossmann, M.; Arnold, E.), s. 657-682 Institutional research plan: CEZ:AV0Z4040901 Keywords : database * nucleic acid Subject RIV: CF - Physical ; Theoretical Chemistry

  1. 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...... strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers...

  2. Peptide Nucleic Acids Complexes of Two Peptide Nucleic Acid Strands and One

    DEFF Research Database (Denmark)

    1999-01-01

    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...

  3. 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....

  4. Nucleic acid delivery with microbubbles and ultrasound.

    Science.gov (United States)

    Rychak, Joshua J; Klibanov, Alexander L

    2014-06-01

    Nucleic acid-based therapy is a growing field of drug delivery research. Although ultrasound has been suggested to enhance transfection decades ago, it took a combination of ultrasound with nucleic acid carrier systems (microbubbles, liposomes, polyplexes, and viral carriers) to achieve reasonable nucleic acid delivery efficacy. Microbubbles serve as foci for local deposition of ultrasound energy near the target cell, and greatly enhance sonoporation. The major advantage of this approach is in the minimal transfection in the non-insonated non-target tissues. Microbubbles can be simply co-administered with the nucleic acid carrier or can be modified to carry nucleic acid themselves. Liposomes with embedded gas or gas precursor particles can also be used to carry nucleic acid, release and deliver it by the ultrasound trigger. Successful testing in a wide variety of animal models (myocardium, solid tumors, skeletal muscle, and pancreas) proves the potential usefulness of this technique for nucleic acid drug delivery. PMID:24486388

  5. Nucleic Acid Aptamers Against Proteases

    OpenAIRE

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø; Andreasen, P A

    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...

  6. Electrochemical analysis of nucleic acids

    Czech Academy of Sciences Publication Activity Database

    Paleček, Emil; Fojta, Miroslav; Jelen, František; Vetterl, Vladimír

    Weinheim : WileyVCH, 2002 - (Bard, A.; Stratmann, M.; Wilson, G.), s. 365-429 ISBN 3-527-30401-0 R&D Projects: GA ČR GV204/97/K084; GA AV ČR IBS5004107; GA AV ČR IAA4004901; GA AV ČR IAA4004002 Institutional research plan: CEZ:AV0Z5004920 Keywords : nucleic acid s * electrochemical analysis * DNA biosensors Subject RIV: BO - Biophysics

  7. Multiplexed microfluidic approach for nucleic acid enrichment

    Energy Technology Data Exchange (ETDEWEB)

    VanderNoot, Victoria A.; Langevin, Stanley Alan; Bent, Zachary; Renzi, Ronald F.; Ferko, Scott M.; Van De Vreugde, James L.; Lane, Todd; Patel, Kamlesh; Branda, Steven

    2016-04-26

    A system for enhancing a nucleic acid sample may include a one pump, a denaturing chamber; a microfluidic hydroxyapatite chromatography device configured for performing hydroxyapatite chromatography on the nucleic acid sample, a sample collector, and tubing connecting the pump with the denaturing chamber, the hydroxyapatite chromatography device and the sample collector such that the pump may be used to move the nucleic acid sample from the denaturing chamber to the hydroxyapatite chromatography device and then to the sample collector.

  8. Applications of radioactively labelled nucleic acid probes

    International Nuclear Information System (INIS)

    Isotopically labelled nucleic acid probes are used extensively in many areas of molecular biology research. Several radioactive isotopes have been utilised for this purpose, with P-32 and S-35 proving the most popular. This contribution will highlight the factors dictating the choice of radioisotope and will describe techniques for in vitro labelling of nucleic acids. The experimental data presented will be focused on applications of labelled nucleic acids including DNA probe assays and DNA sequencing. (author). 9 refs., 2 figs., 4 tabs

  9. 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.......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....

  10. Pulmonary delivery of nucleic acids.

    Science.gov (United States)

    Birchall, James

    2007-11-01

    The lung is an appropriate present and future target for gene therapy approaches designed to treat inherited monogenic diseases, eradicate bronchial tumours, transfer pharmacologically active products to the general circulation, express enzymes to catabolise toxins, manage pulmonary hypertension and lung injury and vaccinate against infection. Despite 35 years of gene therapy research and some significant milestones in molecular biology, the clinical potential of gene therapy has yet to be realised. In pulmonary gene therapy the nucleic acid cargo needs to be delivered to cells in the target region of the lung, and even in cases when these targets are well defined this is severely limited by the pulmonary architecture, clearance mechanisms, immune activation, the presence of respiratory mucus and the availability of a truly representative biological model. The challenge from a drug delivery perspective is to consider the suitability of conventional nebulisers and inhalers for delivering DNA to the lung and design and apply integrated formulation and device solutions specific to nucleic acid delivery. PMID:17970661

  11. Cleaving Double-Stranded DNA with Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    1997-01-01

    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...

  12. Molecular modeling of nucleic acid structure

    OpenAIRE

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

    2001-01-01

    This unit is the first in a series of four units covering the analysis of nucleic acid structure by molecular modeling. This unit provides an overview of computer simulation of nucleic acids. Topics include the static structure model, computational graphics and energy models, generation of an initial model, and characterization of the overall three-dimensional structure.

  13. 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.

  14. Do nucleic acids moonlight as molecular chaperones?

    Science.gov (United States)

    Docter, Brianne E.; Horowitz, Scott; Gray, Michael J.; Jakob, Ursula; Bardwell, James C.A.

    2016-01-01

    Organisms use molecular chaperones to combat the unfolding and aggregation of proteins. While protein chaperones have been widely studied, here we demonstrate that DNA and RNA exhibit potent chaperone activity in vitro. Nucleic acids suppress the aggregation of classic chaperone substrates up to 300-fold more effectively than the protein chaperone GroEL. Additionally, RNA cooperates with the DnaK chaperone system to refold purified luciferase. Our findings reveal a possible new role for nucleic acids within the cell: that nucleic acids directly participate in maintaining proteostasis by preventing protein aggregation. PMID:27105849

  15. NMR studies of nucleic acid dynamics

    Science.gov (United States)

    Al-Hashimi, Hashim M.

    2013-12-01

    Nucleic acid structures have to satisfy two diametrically opposite requirements; on one hand they have to adopt well-defined 3D structures that can be specifically recognized by proteins; on the other hand, their structures must be sufficiently flexible to undergo very large conformational changes that are required during key biochemical processes, including replication, transcription, and translation. How do nucleic acids introduce flexibility into their 3D structure without losing biological specificity? Here, I describe the development and application of NMR spectroscopic techniques in my laboratory for characterizing the dynamic properties of nucleic acids that tightly integrate a broad set of NMR measurements, including residual dipolar couplings, spin relaxation, and relaxation dispersion with sample engineering and computational approaches. This approach allowed us to obtain fundamental new insights into directional flexibility in nucleic acids that enable their structures to change in a very specific functional manner.

  16. Two perspectives to consider Nucleic Acids

    OpenAIRE

    Sosic, A

    2013-01-01

    Since their discovery, nucleic acids have been the object of intense and thorough explorations, leading to the understanding of their structure and functions. Their role as genetic information carriers is well known but there are evidences that they are also involved in a series of other less known processes. No longer have nucleic acids to be considered passive structures, but they are dynamic and active macromolecules, able to assume a number of three-dimensional conformations. Precisely, t...

  17. Rapid nuclear import of short nucleic acids.

    Science.gov (United States)

    Kitagawa, Mai; Okamoto, Akimitsu

    2016-10-01

    Exogenous short-chain nucleic acids undergo rapid import into the nucleus. Fluorescence-labeled dT1-13 DNA microinjected into the cytoplasm domain of a HeLa cell was rapidly imported into the nucleus domain within 1min. This is much more rapid than what has been observed for intracellular diffusion of small molecules. In contrast, import of longer nucleic acids with a length of over 30nt into the nucleus was suppressed. PMID:27597250

  18. Nucleic acid visualization with UCSF Chimera

    OpenAIRE

    Couch, Gregory S.; Hendrix, Donna K.; Ferrin, Thomas E.

    2006-01-01

    With the increase in the number of large, 3D, high-resolution nucleic acid structures, particularly of the 30S and 50S ribosomal subunits and the intact bacterial ribosome, advancements in the visualization of nucleic acid structural features are essential. Large molecular structures are complicated and detailed, and one goal of visualization software is to allow the user to simplify the display of some features and accent others. We describe an extension to the UCSF Chimera molecular visuali...

  19. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of... Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus...

  20. Interaction of Nucleic Acids with the Glycocalyx

    OpenAIRE

    Palte, Michael J.; Raines, Ronald T.

    2012-01-01

    Mammalian cells resist the uptake of nucleic acids. The lipid bilayer of the plasma membrane presents one barrier. Here, we report on a second physicochemical barrier for uptake. To create a sensitive probe for nucleic acid–cell interactions, we synthesized fluorescent conjugates in which lipids are linked to DNA oligonucleotides. We found that these conjugates incorporate readily into the plasma membrane but are not retained there. Expulsion of lipid–oligonucleotide conjugates from the plasm...

  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...

  2. 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.

  3. Detection of nucleic acid sequences by invader-directed cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Brow, Mary Ann D. (Madison, WI); Hall, Jeff Steven Grotelueschen (Madison, WI); Lyamichev, Victor (Madison, WI); Olive, David Michael (Madison, WI); Prudent, James Robert (Madison, WI)

    1999-01-01

    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 5' 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 by charge.

  4. Detection of nucleic acid sequences by invader-directed cleavage

    Science.gov (United States)

    Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

    1999-01-01

    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 5' 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 by charge.

  5. Multifunctional Nucleic Acids for Tumor Cell Treatment

    DEFF Research Database (Denmark)

    Pofahl, Monika; Wengel, Jesper; Mayer, Günter

    2014-01-01

    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......-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...

  6. 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

  7. Chemical consequences of irradiating nucleic acids

    International Nuclear Information System (INIS)

    On the basis of literature data, a discussion is presented of the DNA damage which would be produced in a cellular environment and an attempt is made to place this damage in perspective as a potential hazard in food irradiation. The topics discussed are radiation damage mechanisms, OH reactions with DNA, base products, sugar products, and evaluation of damage from irradiated nucleic acids

  8. 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'-a...

  9. Trends in protein and nucleic acid electroanalysis

    Czech Academy of Sciences Publication Activity Database

    Paleček, Emil

    Seville, 2008. s. 1. [The 59th Annual Meeting of the International Society of Electrochemistry. 07.09.2008-12.09.2008, Seville] Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : protein electroanalysis * nucleic acid electroanalysis Subject RIV: AQ - Safety, Health Protection, Human - Machine

  10. Simulations of nucleic acid vibrational spectra

    Czech Academy of Sciences Publication Activity Database

    Andrushchenko, Valery; Wieser, H.; Bouř, Petr

    Wroclaw : Wroclaw University of Technology, 2010. L24-L24. [Modeling & Design of Molecular Materials 2010. 04.07.2010-08.07.2010, Wroclaw] R&D Projects: GA ČR GAP208/10/0559; GA AV ČR IAA400550702 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleic acid * IR * VCD Subject RIV: CF - Physical ; Theoretical Chemistry

  11. 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...... 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....

  12. Advances in nucleic acid-based detection methods.

    OpenAIRE

    Wolcott, M J

    1992-01-01

    Laboratory techniques based on nucleic acid methods have increased in popularity over the last decade with clinical microbiologists and other laboratory scientists who are concerned with the diagnosis of infectious agents. This increase in popularity is a result primarily of advances made in nucleic acid amplification and detection techniques. Polymerase chain reaction, the original nucleic acid amplification technique, changed the way many people viewed and used nucleic acid techniques in cl...

  13. Advances in magnetofection-magnetically guided nucleic acid delivery

    International Nuclear Information System (INIS)

    Magnetofection is nucleic acid delivery to cells supported and site-specifically guided by the attractive forces of magnetic fields acting on nucleic acid shuttles (vectors) which are associated with magnetic nanoparticles. Recent progress with the method confirms its general applicability with small and large nucleic acids and viruses. The method's therapeutic application as well as mechanistic studies will be discussed

  14. 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...

  15. Detection of nucleic acids by multiple sequential invasive cleavages 02

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Jeff G. (Madison, WI); Lyamichev, Victor I. (Madison, WI); Mast, Andrea L. (Madison, WI); Brow, Mary Ann D. (Madison, WI)

    2002-01-01

    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.

  16. Detection of nucleic acids by multiple sequential invasive cleavages

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Jeff G. (Madison, WI); Lyamichev, Victor I. (Madison, WI); Mast, Andrea L. (Madison, WI); Brow, Mary Ann D. (Madison, WI)

    1999-01-01

    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.

  17. 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.

  18. Novel applications of locked nucleic acids.

    Science.gov (United States)

    Veedu, Rakesh N; Vester, Birte; Wengel, Jesper

    2007-01-01

    Locked Nucleic Acid (LNA) nucleoside triphosphates were prepared and their substrate properties for different polymerases during primer extension and PCR experiments investigated. Phusion High Fidelity DNA polymerase and 9( degrees )Nm(TM) DNA polymerase readily accept LNA nucleoside 5'-triphosphates as substrates in primer extension assays. However, in PCR assays, However, in PCR assays, DNA 9oN(m) polymerase proved to be the best for amplification employing the LNA-A nucleotide. PMID:18029570

  19. Total Nucleic Acid Extraction from Soil

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Roey Angel ### Abstract The following protocol is intended for the simultaneous extraction of DNA and RNA from various soil samples along with suggestions on how to tweak the protocol for soil with higher humic content. The protocol has been used by many and results in very high yields of nucleic acids, typically much more than commercial kits. For buffers and solutions used in this protocol, please see accompanying document Buffers and Solutions for TNA Extractions.pdf. ...

  20. Therapeutic nucleic acids: current clinical status.

    Science.gov (United States)

    Sridharan, Kannan; Gogtay, Nithya Jaideep

    2016-09-01

    Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are simple linear polymers that have been the subject of considerable research in the last two decades and have now moved into the realm of being stand-alone therapeutic agents. Much of this has stemmed from the appreciation that they carry out myriad functions that go beyond mere storage of genetic information and protein synthesis. Therapy with nucleic acids either uses unmodified DNA or RNA or closely related compounds. From both a development and regulatory perspective, they fall somewhere between small molecules and biologics. Several of these compounds are in clinical development and many have received regulatory approval for human use. This review addresses therapeutic uses of DNA based on antisense oligonucleotides, DNA aptamers and gene therapy; and therapeutic uses of RNA including micro RNAs, short interfering RNAs, ribozymes, RNA decoys and circular RNAs. With their specificity, functional diversity and limited toxicity, therapeutic nucleic acids hold enormous promise. However, challenges that need to be addressed include targeted delivery, mass production at low cost, sustaining efficacy and minimizing off-target toxicity. Technological developments will hold the key to this and help accelerate drug approvals in the years to come. PMID:27111518

  1. Extrachromosomal nucleic acids in bovine babesia

    Directory of Open Access Journals (Sweden)

    Isidro Hotzel

    1992-01-01

    Full Text Available Two kinds of small extrachromosomal nucleic acid elements were found in the bovine babesias, Babesia bovis and B. bigemina. One element with an apparent size of 5.5 kilobase pairs (kbp is a double stranded RNA related to virus like particles. Another molecule is a double stranded DNA with a molecular size of about 6.2 kbp. Southern blot comparison of restriction DNA fragments of the latter molecule, which is present in both B. bovis and B. bigemina is described.

  2. A homogeneous nucleic acid hybridization assay based on strand displacement.

    OpenAIRE

    Vary, C P

    1987-01-01

    A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal ...

  3. Microspectrophotometric quantitation of nucleic acid and protein in irradiated epidermis

    International Nuclear Information System (INIS)

    Nucleic acid and proteins of newborn rat tail epidermis subjected to local X-irradiation were microspectrophotometrically studied. Feulgen, gallocyanine chrom-alum and naphthol yellow S methods were performed for demonstration of DNA, total nucleic acid and proteins respectively. The amount of proteins and total nucleic acid increases concomitantly with reactional acanthosis. However, the proteins and nucleic acid decrease as from day 3 post-irradiation. A tentative interpretation of the results would point to a giantization of the epidermic cells not only caused by aqueous imbition but also by an actual increase of the cellular protoplasm. (orig.)

  4. Microspectrophotometric quantitation of nucleic acid and protein in irradiated epidermis.

    Science.gov (United States)

    Conti, C J; Giménez, I B; Cabrini, R L

    1976-03-01

    Nucleic acid and proteins of newborn rat tail subjected to local X-irradiation were microspectrophotometrically studied. Feulgen, gallocyanine chrom-alum and naphthol yellow S methods were performed for demonstration of DNA, total nucleic acid and proteins respectively. The amount of proteins and total nucleic acid increases concomitantly with reactional acanthosis. However, the proteins and nucleic acid decrease as from day 3 post-irradiation. A tentative interpretation of the results would point to a giantization of the epidermic cells not only caused by aqueous imbition but also by an actual increase of the cellular protoplasm. PMID:1258094

  5. Flexibility of nucleic acids: from DNA to RNA

    CERN Document Server

    Bao, Lei; Jin, Lei; Tan, Zhi-Jie

    2015-01-01

    The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs, and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids.

  6. 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

  7. 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.

  8. 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, expr

  9. 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.

  10. Nucleic Acid-Based Nanodevices in Biological Imaging.

    Science.gov (United States)

    Chakraborty, Kasturi; Veetil, Aneesh T; Jaffrey, Samie R; Krishnan, Yamuna

    2016-06-01

    The nanoscale engineering of nucleic acids has led to exciting molecular technologies for high-end biological imaging. The predictable base pairing, high programmability, and superior new chemical and biological methods used to access nucleic acids with diverse lengths and in high purity, coupled with computational tools for their design, have allowed the creation of a stunning diversity of nucleic acid-based nanodevices. Given their biological origin, such synthetic devices have a tremendous capacity to interface with the biological world, and this capacity lies at the heart of several nucleic acid-based technologies that are finding applications in biological systems. We discuss these diverse applications and emphasize the advantage, in terms of physicochemical properties, that the nucleic acid scaffold brings to these contexts. As our ability to engineer this versatile scaffold increases, its applications in structural, cellular, and organismal biology are clearly poised to massively expand. PMID:27294440

  11. Intumescent features of nucleic acids and proteins

    International Nuclear Information System (INIS)

    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/m2). 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/m2, 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

  12. 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.

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

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Wengel, Jesper

    2014-01-01

    '-amino-LNA nucleotides. By application of different chemical reactions, modification of 2'-amino-LNA scaffolds can be efficiently performed in high yields and with various tags, postsynthetically or during the automated oligonucleotide synthesis. The choice of a synthetic method for scaffolding along 2......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......'-amino-LNA mainly 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...

  14. Nucleic Acid Backbone Structure Variations: Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    Nielsen, Peter E.

    2010-01-01

    Synthetic analogues and mimics of the natural genetic material deoxyribonucleic acid (DNA) are potential gene therapeutic (antisense or antigene) drugs. One of these mimics, peptide nucleic acids (PNAs), are chemically closer to peptides and proteins than to DNA, but nonetheless have retained many...... of the structural properties of DNA. These molecules have found applications as probes in genetic diagnostics and are also being developed into antisense (RNA (ribonucleic acid) interference) gene therapeutic drugs, targeting selected genes through sequence-specific recognition of (messenger or micro...

  15. Structural studies of nucleic acids and proteins involved in nucleic acid recognition

    OpenAIRE

    Russo Krauss, Irene

    2010-01-01

    This PhD thesis focuses on the structural analysis of the protein-nucleic acid recognition. In particular the research work has been focalized on two different kinds of proteins and their nucleotide ligands. The first part concerns the structural characterization of complexes between human α-thrombin, a protein of physiological and pathological relevance, and two oligonucleotide aptamers (the so called thrombin binding aptamer and a modified version of it), which adopt a G-quadruplex fold. Th...

  16. 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. PMID:23849929

  17. Computational Approaches to Nucleic Acid Origami.

    Science.gov (United States)

    Jabbari, Hosna; Aminpour, Maral; Montemagno, Carlo

    2015-10-12

    Recent advances in experimental DNA origami have dramatically expanded the horizon of DNA nanotechnology. Complex 3D suprastructures have been designed and developed using DNA origami with applications in biomaterial science, nanomedicine, nanorobotics, and molecular computation. Ribonucleic acid (RNA) origami has recently been realized as a new approach. Similar to DNA, RNA molecules can be designed to form complex 3D structures through complementary base pairings. RNA origami structures are, however, more compact and more thermodynamically stable due to RNA's non-canonical base pairing and tertiary interactions. With all these advantages, the development of RNA origami lags behind DNA origami by a large gap. Furthermore, although computational methods have proven to be effective in designing DNA and RNA origami structures and in their evaluation, advances in computational nucleic acid origami is even more limited. In this paper, we review major milestones in experimental and computational DNA and RNA origami and present current challenges in these fields. We believe collaboration between experimental nanotechnologists and computer scientists are critical for advancing these new research paradigms. PMID:26348196

  18. Nucleic acid detection system and method for detecting influenza

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Molecular Modeling of Nucleic Acid Structure: Energy and Sampling

    OpenAIRE

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

    2001-01-01

    An overview of computer simulation techniques as applied to nucleic acid systems is presented. This unit discusses methods used to treat the energy and to sample representative configurations. Emphasis is placed on molecular mechanics and empirical force fields.

  20. Flexibility of nucleic acids: From DNA to RNA

    Science.gov (United States)

    Lei, Bao; Xi, Zhang; Lei, Jin; Zhi-Jie, Tan

    2016-01-01

    The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs, and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids. Project supported by the National Basic Research Program of China (Grant No. 2011CB933600), the National Natural Science Foundation of China (Grant Nos. 11175132, 11575128, and 11374234), and the Program for New Century Excellent Talents, China (Grant No. NCET 08-0408).

  1. Roles of Intrinsic Disorder in Protein-Nucleic Acid Interactions

    OpenAIRE

    Dyson, H. Jane

    2011-01-01

    Interactions between proteins and nucleic acids typify the role of disordered segments, linkers, tails and other entities in the function of complexes that must form with high affinity and specificity but which must be capable of dissociating when no longer needed. While much of the emphasis in the literature has been on the interactions of disordered proteins with other proteins, disorder is also frequently observed in nucleic acids (particularly RNA) and in the proteins that interact with t...

  2. Nucleic Acid Aptamers: Research Tools in Disease Diagnostics and Therapeutics

    OpenAIRE

    Baby Santosh; Pramod K. Yadava

    2014-01-01

    Aptamers are short sequences of nucleic acid (DNA or RNA) or peptide molecules which adopt a conformation and bind cognate ligands with high affinity and specificity in a manner akin to antibody-antigen interactions. It has been globally acknowledged that aptamers promise a plethora of diagnostic and therapeutic applications. Although use of nucleic acid aptamers as targeted therapeutics or mediators of targeted drug delivery is a relatively new avenue of research, one aptamer-based drug “Ma...

  3. 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....

  4. Hydrophilic magnetic latex for nucleic acid extraction, purification and concentration

    International Nuclear Information System (INIS)

    Core-shell magnetic latex particles bearing poly(N-isopropylacrylamide) in the shell were prepared by encapsulation of magnetic core using a precipitation polymerization process. The cationic character of the particles' surface is favorable for nucleic acid adsorption-desorption by controlling the pH and salinity of the medium. The concentration process of nucleic acids was presented and proven using DNA as a model

  5. Electrical and Electrochemical Monitoring of Nucleic Acid Amplification

    OpenAIRE

    Goda, Tatsuro; Tabata, Miyuki; Miyahara, Yuji

    2015-01-01

    Nucleic acid amplification is a gold standard technique for analyzing a tiny amount of nucleotides in molecular biology, clinical diagnostics, food safety, and environmental testing. Electrical and electrochemical monitoring of the amplification process draws attention over conventional optical methods because of the amenability toward point-of-care applications as there is a growing demand for nucleic acid sensing in situations outside the laboratory. A number of electrical and electrochemic...

  6. Thermodynamics of RNA duplexes modified with unlocked nucleic acid nucleotides

    OpenAIRE

    Pasternak, Anna; Wengel, Jesper

    2010-01-01

    Thermodynamics provides insights into the influence of modified nucleotide residues on stability of nucleic acids and is crucial for designing duplexes with given properties. In this article, we introduce detailed thermodynamic analysis of RNA duplexes modified with unlocked nucleic acid (UNA) nucleotide residues. We investigate UNA single substitutions as well as model mismatch and dangling end effects. UNA residues placed in a central position makes RNA duplex structure less favourable by 4...

  7. Interactions of Night Blue with Nucleic Acids and Determination of Nucleic Acids Using Resonance Light Scattering Technique

    Institute of Scientific and Technical Information of China (English)

    吴会灵; 梁宏; 等

    2003-01-01

    The noncovalent interactions of night blue (NB) with several nucleic acids in buffer medium of Britton-Robinson at pH 4.1 have been studied by spectroscopic methods.It is shown that the binding of NB with nucleic acids involves the J-aggregation of NB molecules on the surface of nucleic acids.The aggregation was encouraged by polyanions nucleic acids,in which nucleic acids served for acting templates,In this connection,a new method of nucleic acids with sensitivity at nanogram level is proposed based on the measurement of enhanced resonance light scattering (RLS).The linear range of ctDNA,fsDNA and yRNA is 0.01-2.5,0.03-2.5 and 0.04-1.0 μg/mL,respectively,and the corresponding detection limits(3σ)are 9.4,7.3 and 5.7ng/mL at 2.5×1005mol/L of NB.Synthetic and real samples were analyzed with satisfactory results.

  8. Nucleic acids in circulation: Are they harmful to the host?

    Indian Academy of Sciences (India)

    Indraneel Mittra; Naveen Kumar Nair; Pradyumna Kumar Mishra

    2012-06-01

    It has been estimated that 1011–1012 cells, primarily of haematogenous origin, die in the adult human body daily, and a similar number is regenerated to maintain homeostasis. Despite the presence of an efficient scavenging system for dead cells, considerable amounts of fragmented genetic material enter the circulation in healthy individuals. Elevated blood levels of extracellular nucleic acids have been reported in various disease conditions; such as ageing and age-related degenerative disorders, cancer; acute and chronic inflammatory conditions, severe trauma and autoimmune disorders. In addition to genomic DNA and nucleosomes, mitochondrial DNA is also found in circulation, as are RNA and microRNA. There is extensive literature that suggests that extraneously added nucleic acids have biological actions. They can enter into cells in vitro and in vivo and induce genetic transformation and cellular and chromosomal damage; and experimentally added nucleic acids are capable of activating both innate and adaptive immune systems and inducing a sterile inflammatory response. The possibility as to whether circulating nucleic acids may, likewise, have biological activities has not been explored. In this review we raise the question as to whether circulating nucleic acids may have damaging effects on the host and be implicated in ageing and diverse acute and chronic human pathologies.

  9. Structural requirements for the procoagulant activity of nucleic acids.

    Directory of Open Access Journals (Sweden)

    Julia Gansler

    Full Text Available Nucleic acids, especially extracellular RNA, are exposed following tissue- or vessel damage and have previously been shown to activate the intrinsic blood coagulation pathway in vitro and in vivo. Yet, no information on structural requirements for the procoagulant activity of nucleic acids is available. A comparison of linear and hairpin-forming RNA- and DNA-oligomers revealed that all tested oligomers forming a stable hairpin structure were protected from degradation in human plasma. In contrast to linear nucleic acids, hairpin forming compounds demonstrated highest procoagulant activities based on the analysis of clotting time in human plasma and in a prekallikrein activation assay. Moreover, the procoagulant activities of the DNA-oligomers correlated well with their binding affinity to high molecular weight kininogen, whereas the binding affinity of all tested oligomers to prekallikrein was low. Furthermore, four DNA-aptamers directed against thrombin, activated protein C, vascular endothelial growth factor and nucleolin as well as the naturally occurring small nucleolar RNA U6snRNA were identified as effective cofactors for prekallikrein auto-activation. Together, we conclude that hairpin-forming nucleic acids are most effective in promoting procoagulant activities, largely mediated by their specific binding to kininogen. Thus, in vivo application of therapeutic nucleic acids like aptamers might have undesired prothrombotic or proinflammatory side effects.

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

    Indian Academy of Sciences (India)

    Wing-Fu Lai

    2011-09-01

    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 into action, and it is the latter that has been far from adequately addressed at the moment. This was demonstrated by a database search on PubMed and Web of Science, from which only seven studies published between 2000 and 2010 were found to directly touch on the development of nucleic acid therapy for anti-aging and/or longevity enhancing purposes. In light of this, the objective of this article is to overview the current understanding of the intimate association between genes and longevity, and to bring the prospect of nucleic acid therapy for lifespan prolongation to light.

  11. Prospects for using self-assembled nucleic acid structures.

    Science.gov (United States)

    Rudchenko, M N; Zamyatnin, A A

    2015-04-01

    According to the central dogma in molecular biology, nucleic acids are assigned with key functions on storing and executing genetic information in any living cell. However, features of nucleic acids are not limited only with properties providing template-dependent biosynthetic processes. Studies of DNA and RNA unveiled unique features of these polymers able to make various self-assembled three-dimensional structures that, among other things, use the complementarity principle. Here, we review various self-assembled nucleic acid structures as well as application of DNA and RNA to develop nanomaterials, molecular automata, and nanodevices. It can be expected that in the near future results of these developments will allow designing novel next-generation diagnostic systems and medicinal drugs. PMID:25869355

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

    International Nuclear Information System (INIS)

    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

  13. 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; Filichev, Vyacheslav V

    2011-01-01

    Two G-quadruplex forming sequences, 50-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4-(1-pyrenylet......Two G-quadruplex forming sequences, 50-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4......-(1-pyrenylethynyl)phenylmethyl]glycerol (twisted intercalating nucleic acid, TINA). Incorporation of LNA or INA/TINA monomers provide as much as 8-fold improvement of anti-HIV-1 activity. We demonstrate for the first time a detailed analysis of the effect the incorporation of INA/TINA monomers in quadruplex forming...... of the 50-phosphate inhibits dimerisation of this G-quadruplex as a result of negative charge–charge repulsion. Contrary to that, we found that attachment of the 50-O-DMT-group produced a more active 17-mer sequence that showed signs of aggregation—forming multimeric G-quadruplex species in solution...

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

    DEFF Research Database (Denmark)

    Pasternak, Anna; Wengel, Jesper

    2011-01-01

    DNA duplexes, quadruplexes or i-motifs. Moreover, UNA monomers were found to be compatible with RNase H activity, a property which is important for single stranded antisense constructs. Notably, UNA monomers can be applied in the design of superior siRNAs, combining potent gene silencing and......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...

  15. Redox labeling and redox coding of nucleic acids

    Czech Academy of Sciences Publication Activity Database

    Balintová, Jana; Brázdová, Marie; Havran, Luděk; Fojta, Miroslav; Hocek, Michal

    Praha: Institute of Organic Chemistry and Biochemistry AS CR, v. v. i, 2014 - (Hocek, M.), s. 229-230. (Collection Symposium Series. 14). ISBN 978-80-86241-50-0. [Symposium on Chemistry of Nucleic Acid Components /16./. Český Krumlov (CZ), 08.06.2014-13.06.2014] R&D Projects: GA ČR GBP206/12/G151; GA AV ČR(CZ) IAA400040901 Institutional support: RVO:61388963 ; RVO:68081707 Keywords : nucleic acids * DNA * dNTPs Subject RIV: CC - Organic Chemistry; BO - Biophysics (BFU-R)

  16. Spherical Nucleic Acids as Intracellular Agents for Nucleic Acid Based Therapeutics

    Science.gov (United States)

    Hao, Liangliang

    Recent functional discoveries on the noncoding sequences of human genome and transcriptome could lead to revolutionary treatment modalities because the noncoding RNAs (ncRNAs) can be applied as therapeutic agents to manipulate disease-causing genes. To date few nucleic acid-based therapeutics have been translated into the clinic due to challenges in the delivery of the oligonucleotide agents in an effective, cell specific, and non-toxic fashion. Unmodified oligonucleotide agents are destroyed rapidly in biological fluids by enzymatic degradation and have difficulty crossing the plasma membrane without the aid of transfection reagents, which often cause inflammatory, cytotoxic, or immunogenic side effects. Spherical nucleic acids (SNAs), nanoparticles consisting of densely organized and highly oriented oligonucleotides, pose one possible solution to circumventing these problems in both the antisense and RNA interference (RNAi) pathways. The unique three dimensional architecture of SNAs protects the bioactive oligonucleotides from unspecific degradation during delivery and supports their targeting of class A scavenger receptors and endocytosis via a lipid-raft-dependent, caveolae-mediated pathway. Owing to their unique structure, SNAs are able to cross cell membranes and regulate target genes expression as a single entity, without triggering the cellular innate immune response. Herein, my thesis has focused on understanding the interactions between SNAs and cellular components and developing SNA-based nanostructures to improve therapeutic capabilities. Specifically, I developed a novel SNA-based, nanoscale agent for delivery of therapeutic oligonucleotides to manipulate microRNAs (miRNAs), the endogenous post-transcriptional gene regulators. I investigated the role of SNAs involving miRNAs in anti-cancer or anti-inflammation responses in cells and in in vivo murine disease models via systemic injection. Furthermore, I explored using different strategies to construct

  17. Delivery Systems for In Vivo use of Nucleic Acid Drugs

    Directory of Open Access Journals (Sweden)

    Resende R.R

    2007-01-01

    Full Text Available The notorious biotechnological advance of the last few decades has allowed the development of experimental methods for understanding molecular mechanisms of genes and new therapeutic approaches. Gene therapy is maturing into a viable, practical method with the potential to cure a variety of human illnesses. Some nucleic-acid-based drugs are now available for controlling the progression of genetic diseases by inhibiting gene expression or the activity of their gene products. New therapeutic strategies employ a wide range of molecular tools such as bacterial plasmids containing transgenic inserts, RNA interference aptamers. A nucleic-acid based constitution confers a lower immunogenic potential and as result of the high stringency selection of large molecular variety, these drugs have high affi nity and selectivity for their targets. However, nucleic acids have poor biostability thus requiring chemical modifications and delivery systems to maintain their activity and ease their cellular internalization. This review discusses some of the mechanisms of action and the application of therapies based on nucleic acids such as aptamers and RNA interference as well as platforms for cellular uptake and intracellular delivery of therapeutic oligonucleotides and their trade-offs.

  18. Arrays of nucleic acid probes on biological chips

    Science.gov (United States)

    Chee, Mark; Cronin, Maureen T.; Fodor, Stephen P. A.; Huang, Xiaohua X.; Hubbell, Earl A.; Lipshutz, Robert J.; Lobban, Peter E.; Morris, MacDonald S.; Sheldon, Edward L.

    1998-11-17

    DNA chips containing arrays of oligonucleotide probes can be used to determine whether a target nucleic acid has a nucleotide sequence identical to or different from a specific reference sequence. The array of probes comprises probes exactly complementary to the reference sequence, as well as probes that differ by one or more bases from the exactly complementary probes.

  19. Mutagenesis and carcinogenesis caused by the oxidation of nucleic acids.

    OpenAIRE

    Nakabeppu, Yusaku; Sakumi, Kunihiko; Sakamoto, Katsumi; Tsuchimoto, Daisuke; Tsuzuki, Teruhisa; Nakatsu, Yoshimichi

    2006-01-01

    KEYWORDS CLASSIFICATION: adverse effects;Animals;chemistry;deficiency;DNA Damage;DNA Glycosylases;DNA Repair;DNA Repair Enzymes;Enzymes;genetics;Genomics;Guanine;Hydrolases;Intestinal Neoplasms;Japan;Liver Neoplasms;metabolism;mechanisms of carcinogenesis;Mice;Mutagenesis;Mutation;Neoplasms;Nucleic Acids;Oxidation-Reduction;Oxidative Stress;Phosphoric Monoester Hydrolases;Skin Neoplasms;Ultraviolet Rays.

  20. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. 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

    2016-08-09

    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.

  3. Circulating nucleic acids as a new diagnostic tool

    Czech Academy of Sciences Publication Activity Database

    Urbanová, Markéta; Plzák, J.; Strnad, Hynek; Betka, J.

    2010-01-01

    Roč. 15, č. 2 (2010), s. 242-259. ISSN 1425-8153 R&D Projects: GA MŠk 2B06106 Institutional research plan: CEZ:AV0Z50520514 Keywords : circulating nucleic acids * diagnostics * cancer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.455, year: 2010

  4. 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

  5. "Clickable" LNA/DNA probes for fluorescence sensing of nucleic acids and autoimmune antibodies

    DEFF Research Database (Denmark)

    Jørgensen, Anna S; Gupta, Pankaj; Wengel, Jesper; Astakhova, I Kira

    2013-01-01

    Herein we describe fluorescent oligonucleotides prepared by click chemistry between novel alkyne-modified locked nucleic acid (LNA) strands and a series of fluorescent azides for homogeneous (all-in-solution) detection of nucleic acids and autoimmune antibodies.......Herein we describe fluorescent oligonucleotides prepared by click chemistry between novel alkyne-modified locked nucleic acid (LNA) strands and a series of fluorescent azides for homogeneous (all-in-solution) detection of nucleic acids and autoimmune antibodies....

  6. A real-time assay for monitoring nucleic acid cleavage by quadruplex formation

    OpenAIRE

    Kankia, Besik I.

    2006-01-01

    Direct and straightforward methods to follow nucleic acid cleavage are needed. A spectrophotometric quadruplex formation assay (QFA) was developed, which allows real-time monitoring of site-specific cleavage of nucleic acids. QFA was applied to study both protein and nucleic acid restriction enzymes, and was demonstrated to accurately determine Michaelis–Menten parameters for the cleavage reaction catalyzed by EcoRI. QFA can be used to study the mechanisms of protein–nucleic acid recognition....

  7. Peptide nucleic acids and their potential applications in biotechnology

    DEFF Research Database (Denmark)

    Buchardt, O.; Egholm, M.; Berg, R.H.; Nielsen, P.E.

    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 and...... protease attack in serum and cellular extracts and, thus, appear very promising as diagnostic and biomolecular probes, and possibly as antisense and antigene drugs....

  8. Nucleic Acid Charge Transfer: Black, White and Gray

    OpenAIRE

    Venkatramani, Ravindra; Keinan, Shahar; Balaeff, Alexander; Beratan, David N.

    2011-01-01

    Theoretical studies of charge transport in deoxyribonucleic acid (DNA) and peptide nucleic acid (PNA) indicate that structure and dynamics modulate the charge transfer rates, and that different members of a structural ensemble support different charge transport mechanisms. Here, we review the influences of nucleobase geometry, electronic structure, solvent environment, and thermal conformational fluctuations on the charge transfer mechanism. We describe an emerging framework for understanding...

  9. 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; Pedersen, Erik B

    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...

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

    Lifescience Database Archive (English)

    Full Text Available 17082566 Nucleic acid-sensing TLRs as modifiers of autoimmunity. Deane JA, Bolland ...S. J Immunol. 2006 Nov 15;177(10):6573-8. (.png) (.svg) (.html) (.csml) Show Nucleic acid-sensing TLRs as modifiers of autoimmunity.... PubmedID 17082566 Title Nucleic acid-sensing TLRs as modifiers of autoimmunity. Aut

  11. Nucleic acids encoding antifungal polypeptides and uses thereof

    Science.gov (United States)

    Altier, Daniel J.; Ellanskaya, I. A.; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2010-11-02

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include an amino acid sequence, and variants and fragments thereof, for an antipathogenic polypeptide that was isolated from a fungal fermentation broth. Nucleic acid molecules that encode the antipathogenic polypeptides of the invention, and antipathogenic domains thereof, are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention are also disclosed.

  12. 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.

  13. Nucleic acid-based approaches to STAT inhibition.

    Science.gov (United States)

    Sen, Malabika; Grandis, Jennifer R

    2012-10-01

    Silencing of abnormally activated genes can be accomplished in a highly specific manner using nucleic acid based approaches. The focus of this review includes the different nucleic acid based inhibition strategies such as antisense oligodeoxynucleotides, small interfering RNA (siRNA), dominant-negative constructs, G-quartet oligonucleotides and decoy oligonucleotides, their mechanism of action and the effectiveness of these approaches to targeting the STAT (signal transducer and activator of transcription) proteins in cancer. Among the STAT proteins, especially STAT3, followed by STAT5, are the most frequently activated oncogenic STATs, which have emerged as plausible therapeutic cancer targets. Both STAT3 and STAT5 have been shown to regulate numerous oncogenic signaling pathways including proliferation, survival, angiogenesis and migration/invasion. PMID:24058785

  14. Nucleic acid detection in the diagnosis and prevention of schistosomiasis.

    Science.gov (United States)

    He, Ping; Song, Lan-Gui; Xie, Hui; Liang, Jin-Yi; Yuan, Dong-Ya; Wu, Zhong-Dao; Lv, Zhi-Yue

    2016-01-01

    Schistosomiasis is an important zoonotic parasitic disease that causes serious harms to humans and animals. Surveillance and diagnosis play key roles in schistosomiasis control, however, current techniques for surveillance and diagnosis of the disease have limitations. As genome data for parasites are increasing, novel techniques for detection incorporating nucleotide sequences are receiving widespread attention. These sensitive, specific, and rapid detection methods are particularly important in the diagnosis of low-grade and early infections, and may prove to have clinical significance. This paper reviews the progress of nucleic acid detection in the diagnosis and prevention of schistosomiasis, including such aspects as the selection of target genes, and development and application of nucleic acid detection methods. PMID:27025210

  15. Clinical applications of nucleic acid aptamers in cancer

    OpenAIRE

    PEI, XIAOYU; Jun ZHANG; Liu, Jie

    2014-01-01

    Nucleic acid aptamers are small single-stranded DNA or RNA oligonucleotide segments, which bind to their targets with high affinity and specificity via unique three-dimensional structures. Aptamers are generated by an iterative in vitro selection process, termed as systematic evolution of ligands by exponential enrichment. Owing to their specificity, non-immunogenicity, non-toxicity, easily modified chemical structure and wide range of targets, aptamers appear to be ideal candidates for vario...

  16. Nucleic Acid Aptamers for Target Validation and Therapeutic Applications

    OpenAIRE

    Pendergrast, P. Shannon; Marsh, H Nicholas; Grate, Dilara; Healy, Judith M.; Stanton, Martin

    2005-01-01

    In the simplest view, aptamers can be thought of as nucleic acid analogs to antibodies. They are able to bind specifically to proteins, and, in many cases, that binding leads to a modulation of protein activity. New aptamers are rapidly generated through the SELEX (Systematic Evolution of Ligands by Exponential enrichment) process and have a very high target affinity and specificity (picomoles to nanomoles). Furthermore, aptamers composed of modified nucleotides have a long in vivo half-life ...

  17. Nucleic acid aptamers: clinical applications and promising new horizons

    OpenAIRE

    Ni, Xiaohua; Castanares, Mark; Mukherjee, Amarnath; Shawn E Lupold

    2011-01-01

    Aptamers are a special class of nucleic acid molecules that are beginning to be investigated for clinical use. These small RNA/DNA molecules can form secondary and tertiary structures capable of specifically binding proteins or other cellular targets; they are essentially a chemical equivalent of antibodies. Aptamers have the advantage of being highly specific, relatively small in size, and non-immunogenic. Since the discovery of aptamers in the early 1990s, great efforts have been made to ma...

  18. Solid-state NMR studies of nucleic acid components

    Czech Academy of Sciences Publication Activity Database

    Dračínský, Martin; Hodgkinson, P.

    2015-01-01

    Roč. 5, č. 16 (2015), s. 12300-12310. ISSN 2046-2069 R&D Projects: GA ČR GA13-24880S Institutional support: RVO:61388963 Keywords : NMR spectroscopy * nucleic acids * solid-state NMR Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.840, year: 2014 http://pubs.rsc.org/en/content/articlepdf/2015/ra/c4ra14404j

  19. Mass spectral characterization of a protein-nucleic acid photocrosslink.

    OpenAIRE

    Golden, M. C.; Resing, K. A.; Collins, B. D.; Willis, M. C.; Koch, T H

    1999-01-01

    A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with ...

  20. Biomimetic High Density Lipoprotein Nanoparticles For Nucleic Acid Delivery

    OpenAIRE

    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

    2011-01-01

    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, t...

  1. Delivery Systems for In Vivo use of Nucleic Acid Drugs

    OpenAIRE

    Resende R.R; Torres H.A.M; Yuahasi K.K; Majumder P; Ulrich H.

    2007-01-01

    The notorious biotechnological advance of the last few decades has allowed the development of experimental methods for understanding molecular mechanisms of genes and new therapeutic approaches. Gene therapy is maturing into a viable, practical method with the potential to cure a variety of human illnesses. Some nucleic-acid-based drugs are now available for controlling the progression of genetic diseases by inhibiting gene expression or the activity of their gene products. New therapeutic st...

  2. Non-natural Nucleic Acids for Synthetic Biology

    OpenAIRE

    Appella, Daniel H.

    2009-01-01

    Genetic manipulation is an important facet of synthetic biology but can be complicated by undesired nuclease degradation. Incorporating non-natural nucleic acids into a gene could convey resistance to nucleases and promote expression. The compatibility of non-natural nucleosides with polymerases is reviewed with a focus on results from the past two years. Details are provided about how the different systems could be useful in synthetic biology.

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

    OpenAIRE

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

    2015-01-01

    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 (poly...

  4. New Approaches Towards Recognition of Nucleic Acid Triple Helices

    OpenAIRE

    Arya, Dev P.

    2010-01-01

    We show that groove recognition of nucleic acid triple helices can be achieved with aminosugars. Among these aminosugars, neomycin is the most effective aminoglycoside (groove binder) for stabilizing a DNA triple helix. It stabilizes both the T·A·T triplex and mixed-base DNA triplexes better than known DNA minor groove binders (which usually destabilize the triplex) and polyamines. Neomycin selectively stabilizes the triplex (T·A·T and mixed base) without any effect on the DNA duplex. The sel...

  5. Intracellular Fate of Spherical Nucleic Acid Nanoparticle Conjugates

    OpenAIRE

    Wu, Xiaochen A.; Choi, Chung Hang J.; Zhang, Chuan; Hao, Liangliang; Mirkin, Chad A.

    2014-01-01

    Spherical nucleic acid (SNA) nanoparticle conjugates are a class of bionanomaterials that are extremely potent in many biomedical applications. Their unique ability to enter multiple mammalian cell types as single-entity agents arises from their novel three-dimensional architecture, which consists of a dense shell of highly oriented oligonucleotides chemically attached typically to a gold nanoparticle core. This architecture allows SNAs to engage certain cell surface receptors to facilitate e...

  6. 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...

  7. 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

  8. The Protein and Nucleic Acid (PAN) Facility at Stanford University

    OpenAIRE

    Eckart, M.; Kosovilka, N.; Sanchez, A; Tran, Y; Walker, P; Winant, R.; Zuo, E.; Patel, S.

    2010-01-01

    The Protein and Nucleic Acid (PAN) Facility (http://pan.stanford.edu) at Stanford University's Beckman Center is a multifaceted biotechnology fee-for-service laboratory providing services to the Stanford scientific community, other non-profit and biopharmaceutical organizations. The Facility's mission is to be adaptable and responsive to the changing needs of biomedical research by providing basic science investigators continued access to key tools and applications in an efficient and cost ef...

  9. System for portable nucleic acid testing in low resource settings

    Science.gov (United States)

    Lu, Hsiang-Wei; Roskos, Kristina; Hickerson, Anna I.; Carey, Thomas; Niemz, Angelika

    2013-03-01

    Our overall goal is to enable timely diagnosis of infectious diseases through nucleic acid testing at the point-of-care and in low resource settings, via a compact system that integrates nucleic acid sample preparation, isothermal DNA amplification, and nucleic acid lateral flow (NALF) detection. We herein present an interim milestone, the design of the amplification and detection subsystem, and the characterization of thermal and fluidic control and assay execution within this system. Using an earlier prototype of the amplification and detection unit, comprised of a disposable cartridge containing flexible pouches, passive valves, and electrolysis-driven pumps, in conjunction with a small heater, we have demonstrated successful execution of an established and clinically validated isothermal loop-mediated amplification (LAMP) reaction targeting Mycobacterium tuberculosis (M.tb) DNA, coupled to NALF detection. The refined design presented herein incorporates miniaturized and integrated electrolytic pumps, novel passive valves, overall design changes to facilitate integration with an upstream sample preparation unit, and a refined instrument design that automates pumping, heating, and timing. Nucleic acid amplification occurs in a two-layer pouch that facilitates fluid handling and appropriate thermal control. The disposable cartridge is manufactured using low-cost and scalable techniques and forms a closed system to prevent workplace contamination by amplicons. In a parallel effort, we are developing a sample preparation unit based on similar design principles, which performs mechanical lysis of mycobacteria and DNA extraction from liquefied and disinfected sputum. Our next step is to combine sample preparation, amplification, and detection in a final integrated cartridge and device, to enable fully automated sample-in to answer-out diagnosis of active tuberculosis in primary care facilities of low-resource and high-burden countries.

  10. Peptide nucleic acid - an opportunity for bio-nanotechnology

    OpenAIRE

    Anstätt, Philipp; Gasser, Gilles

    2014-01-01

    DNA is a major player in the field of bio-nanotechnology and many interesting applications have been realized using this oligonucleotide. In contrast, the use of peptide nucleic acid (PNA), which is a non-natural, neutral analogue of DNA with superior hybridization strengths compared to DNA, is still in its infancy in bio-nanotechnology. However, as demonstrated in this short review using selected studies, promising examples demonstrating the tremendous opportunities that PNA can offer for bi...

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

    OpenAIRE

    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 sensit...

  12. Extracellular Nucleic Acids in Blood of Patients with Chronic Obstructive Pulmonary Disease

    OpenAIRE

    Larissa E. Muravlyova; Vilen B. Molotov-Luchanskiy; Dmitriy A. Klyuyev; Ryszhan E. Bakirova; Ludmila A. Demidchik; Evgeniya A. Kolesnikova

    2013-01-01

    The concentrations of extracellular nucleic acids and acid-soluble precursors of nucleic acids in blood of patients with different forms and severity of chronic obstructive pulmonary disease (COPD) were evaluated. The significant increase of the content of extracellular RNA and acid-soluble precursors of nucleic acids in plasma of patients with COPD was detected. The decrease of extracellular RNA in plasma of patients with COPD worsening was diagnosed. Extracellular DNA in plasma and red bloo...

  13. Glycol methacrylate in light microscopy: nucleic acid cytochemistry.

    Science.gov (United States)

    Cole, M B; Ellinger, J

    1981-07-01

    Techniques utilizing Feulgen, azure B bromide, methyl green-pyronin, gallocyanin chromalum and cresyl violet stains have been modified and adapted for visualizing nucleic acids in 0.5-2.0 micrometer sections of tissues embedded in glycol methacrylate (GMA). Methods for evaluating the stain specificity for DNA and RNA using deoxyribonuclease and ribonuclease digestions, aldehyde blocking, and acid extractions are also described. The specificity of the stains in GMA embedded tissues is comparable to that reported for paraffin-embedded tissues. PMID:6167720

  14. Orientation Preferences of Backbone Secondary Amide Functional Groups in Peptide Nucleic Acid Complexes: Quantum Chemical Calculations Reveal an Intrinsic Preference of Cationic D-Amino Acid-Based Chiral PNA Analogues for the P-form

    OpenAIRE

    Topham, Christopher M.; Smith, Jeremy C.

    2006-01-01

    Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like basepair stacking. Quantum chemical calculations on the bindin...

  15. 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.

  16. Non-enzymatic depurination of nucleic acids: factors and mechanisms.

    Science.gov (United States)

    An, Ran; Jia, Yu; Wan, Baihui; Zhang, Yanfang; Dong, Ping; Li, Jing; Liang, Xingguo

    2014-01-01

    Depurination has attracted considerable attention since a long time for it is closely related to the damage and repair of nucleic acids. In the present study, depurination using a pool of 30-nt short DNA pieces with various sequences at diverse pH values was analyzed by High Performance Liquid Chromatography (HPLC). Kinetic analysis results showed that non-enzymatic depurination of oligodeoxynucleotides exhibited typical first-order kinetics, and its temperature dependence obeyed Arrhenius' law very well. Our results also clearly showed that the linear relationship between the logarithms of rate constants and pH values had a salient point around pH 2.5. Interestingly and unexpectedly, depurination depended greatly on the DNA sequences. The depurination of poly (dA) was found to be extremely slow, and thymine rich sequences depurinated faster than other sequences. These results could be explained to some extent by the protonation of nucleotide bases. Moreover, two equations were obtained based on our data for predicting the rate of depurination under various conditions. These results provide basic data for gene mutagenesis and nucleic acids metabolism in acidic gastric juice and some acidic organelles, and may also help to rectify some misconceptions about depurination. PMID:25546310

  17. Non-enzymatic depurination of nucleic acids: factors and mechanisms.

    Directory of Open Access Journals (Sweden)

    Ran An

    Full Text Available Depurination has attracted considerable attention since a long time for it is closely related to the damage and repair of nucleic acids. In the present study, depurination using a pool of 30-nt short DNA pieces with various sequences at diverse pH values was analyzed by High Performance Liquid Chromatography (HPLC. Kinetic analysis results showed that non-enzymatic depurination of oligodeoxynucleotides exhibited typical first-order kinetics, and its temperature dependence obeyed Arrhenius' law very well. Our results also clearly showed that the linear relationship between the logarithms of rate constants and pH values had a salient point around pH 2.5. Interestingly and unexpectedly, depurination depended greatly on the DNA sequences. The depurination of poly (dA was found to be extremely slow, and thymine rich sequences depurinated faster than other sequences. These results could be explained to some extent by the protonation of nucleotide bases. Moreover, two equations were obtained based on our data for predicting the rate of depurination under various conditions. These results provide basic data for gene mutagenesis and nucleic acids metabolism in acidic gastric juice and some acidic organelles, and may also help to rectify some misconceptions about depurination.

  18. Practical application of nucleic acid techniques to avian disease problems.

    Science.gov (United States)

    Purchase, H G

    1989-01-01

    A workshop in which 17 practicing scientists participated was intended to address primarily people who use or could use biotechnology in their work and was confined to five techniques. Endonuclease fingerprinting and mapping involved cleaving nucleic acid with a specific restriction enzyme and separating the nucleic acid fragments by electrophoresis. Field and vaccine isolates of Pasteurella multocida could be distinguished; Salmonella enteritidis could be divided into three groups; chlamydia could be grouped into seven groups; and vaccinia, quail pox, and fowl pox could be clearly distinguished. Preparation of nucleic acid probes involved producing large amounts of labeled oligonucleotides, usually of unknown sequence. Successful probes had been made for infectious bursal disease virus, avian influenza virus, Newcastle disease virus, and infectious bronchitis virus. In Southern, Northern, and dot blotting, either DNA or RNA fragments were placed on or transferred to a solid substrate and probed. The procedure was able to detect infectious bursal disease virus, infectious bronchitis virus, Mycoplasma gallisepticum, and Marek's disease virus. In situ hybridization involved applying a labeled probe to frozen or fixed sections or to intact cells. In Polymerase chain reaction, two primers, some distance apart, were annealed to a denatured target DNA. Repeated cycles of DNA synthesis with a thermostable polymerase, denaturing, and reannealing resulted in great amplification of a rare sequence. After 30 cycles, a rare gene sequence could be amplified more than 10(6) times. It was used successfully to detect minute quantities of influenza virus and infectious bursal disease virus, and the process was used to facilitate DNA sequencing of coccidiosis gene segments. PMID:2559697

  19. Solving nucleic acid structures by molecular replacement: examples from group II intron studies

    International Nuclear Information System (INIS)

    Strategies for phasing nucleic acid structures by molecular replacement, using both experimental and de novo designed models, are discussed. Structured RNA molecules are key players in ensuring cellular viability. It is now emerging that, like proteins, the functions of many nucleic acids are dictated by their tertiary folds. At the same time, the number of known crystal structures of nucleic acids is also increasing rapidly. In this context, molecular replacement will become an increasingly useful technique for phasing nucleic acid crystallographic data in the near future. Here, strategies to select, create and refine molecular-replacement search models for nucleic acids are discussed. Using examples taken primarily from research on group II introns, it is shown that nucleic acids are amenable to different and potentially more flexible and sophisticated molecular-replacement searches than proteins. These observations specifically aim to encourage future crystallographic studies on the newly discovered repertoire of noncoding transcripts

  20. 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.

  1. Solving nucleic acid structures by molecular replacement: examples from group II intron studies

    Energy Technology Data Exchange (ETDEWEB)

    Marcia, Marco, E-mail: marco.marcia@yale.edu; Humphris-Narayanan, Elisabeth; Keating, Kevin S.; Somarowthu, Srinivas [Yale University, New Haven, CT 06511 (United States); Rajashankar, Kanagalaghatta [Argonne National Laboratory, Argonne, IL 60439 (United States); Pyle, Anna Marie, E-mail: marco.marcia@yale.edu [Yale University, New Haven, CT 06511 (United States); Yale University, New Haven, CT 06511 (United States); Howard Hughes Medical Institute, Chevy Chase, MD 20815 (United States)

    2013-11-01

    Strategies for phasing nucleic acid structures by molecular replacement, using both experimental and de novo designed models, are discussed. Structured RNA molecules are key players in ensuring cellular viability. It is now emerging that, like proteins, the functions of many nucleic acids are dictated by their tertiary folds. At the same time, the number of known crystal structures of nucleic acids is also increasing rapidly. In this context, molecular replacement will become an increasingly useful technique for phasing nucleic acid crystallographic data in the near future. Here, strategies to select, create and refine molecular-replacement search models for nucleic acids are discussed. Using examples taken primarily from research on group II introns, it is shown that nucleic acids are amenable to different and potentially more flexible and sophisticated molecular-replacement searches than proteins. These observations specifically aim to encourage future crystallographic studies on the newly discovered repertoire of noncoding transcripts.

  2. Metallophilic HgII...HgII interactions in nucleic acids

    Czech Academy of Sciences Publication Activity Database

    Benda, Ladislav; Tanaka, Y.; Sychrovský, Vladimír; Straka, Michal

    Praha: Matfyzpress, 2011 - (Burda, J.). s. 62-62 ISBN 978-80-7378-180-4. [Modeling Interactions in Biomolecules /5./. 04.09.2011-09.09.2011, Kutná Hora] R&D Projects: GA ČR GA203/09/2037; GA ČR GAP205/10/0228 Grant ostatní: European Reintegration Grant(XE) 230955 Institutional research plan: CEZ:AV0Z40550506 Keywords : metallophilic HgII...HgII interactions * metallophilic interactions * base stacking * nucleic acids Subject RIV: CF - Physical ; Theoretical Chemistry

  3. 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...

  4. Affinity sorbents containing nucleic acids and their fragments

    International Nuclear Information System (INIS)

    The published data on the main methods for the preparation of polymeric supports containing nucleic acids (NA) or their fragments (oligonucleotides) are reviewed with special emphasis on chemical immobilisation. Some physical and physicochemical immobilisation techniques, including those based on the use of enzymes and avidin-biotin interactions and preparation of NA-containing supports by direct oligonucleotide synthesis on these supports are considered. A special section is devoted to the application of NA-containing sorbents for the isolation of individual NA and proteins as well as in hybridisation analysis including those utilising DNA chips and DNA biosensors. The bibliography includes 391 references.

  5. Structure and function analysis of protein–nucleic acid complexes

    Science.gov (United States)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein–nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

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

    OpenAIRE

    Heemstra, Jennifer M.; Liu, David Ruchien

    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...

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

    OpenAIRE

    Wai-Leng Lee; Jing-Ying Huang; Lie-Fen Shyur

    2013-01-01

    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 th...

  8. Ionizing radiation induced attachment reactions of nucleic acids and their components

    International Nuclear Information System (INIS)

    An extensive bibliographic review is given of experimental and theoretical data on radiation-induced attachment reactions of nucleic acids and their components. Mechanisms of these reactions are reviewed. The reactions with water, formate, and alcohols, with amines and other small molecules, and with radiation sensitizers and nucleic acid-nucleic acid reactions are discussed. Studies of the reaction mechanisms show that many of the reactions occur by radical-molecule reactions, but radical-radical reactions also occur. Radiation modifiers become attached to nucleic acids in vitro and in vivo and there are indications that attachment may be necessary for the action of some sensitizers. (U.S.)

  9. Nucleic acid probes in the diagnosis of plant viruses and viroids

    International Nuclear Information System (INIS)

    This paper presents an approach to the diagnosis of plant pathogens which utilizes the technique of nucleic acid hybridization. A nucleic acid probe, either labeled with a radioactive isotope or labeled nonisotopically, hybridizes to form a duplex with a target nucleic acid which is exactly complementary to itself. No such double-strand hybrid is formed with other nucleic acids. The specificity, sensitivity, and speed of molecular hybridization allows the method to be valuable adjunct to the more conventional immunological approaches used in diagnostic plant virology

  10. Interaction of nucleic acids with carbon nanotubes and dendrimers

    Indian Academy of Sciences (India)

    Bidisha Nandy; Mogurampelly Santosh; Prabal K Maiti

    2012-07-01

    Nucleic acid interaction with nanoscale objects like carbon nanotubes (CNTs) and dendrimers is of fundamental interest because of their potential application in CNT separation, gene therapy and antisense therapy. Combining nucleic acids with CNTs and dendrimers also opens the door towards controllable self-assembly to generate various supra-molecular and nano-structures with desired morphologies. The interaction between these nanoscale objects also serve as a model system for studying DNA compaction, which is a fundamental process in chromatin organization. By using fully atomistic simulations, here we report various aspects of the interactions and binding modes of DNA and small interfering RNA (siRNA) with CNTs, graphene and dendrimers. Our results give a microscopic picture and mechanism of the adsorption of single- and double-strand DNA (ssDNA and dsDNA) on CNT and graphene. The nucleic acid–CNT interaction is dominated by the dispersive van der Waals (vdW) interaction. In contrast, the complexation of DNA (both ssDNA and dsDNA) and siRNA with various generations of poly-amido-amine (PAMAM) dendrimers is governed by electrostatic interactions. Our results reveal that both the DNA and siRNA form stable complex with the PAMAM dendrimer at a physiological pH when the dendrimer is positively charged due to the protonation of the primary amines. The size and binding energy of the complex increase with increase in dendrimer generation. We also give a summary of the current status in these fields and discuss future prospects.

  11. Synthesis of New Chrial Building Blocks for Novel Peptide Nucleic Acids

    Institute of Scientific and Technical Information of China (English)

    WU,Jie; XU,Xiao-Yu; LIU,Ke-Liang

    2003-01-01

    N-Boc protected amino acids of analogues of peptide nucleic acid (PNA),which are a class of conformationally constrained building blocks based on 4-aminoproline backbone with chirality at 2-c and 4-c,have been synthesized.Those monomers can be used for the construction of novel peptide nucleic acid analogues.

  12. 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.

  13. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    Science.gov (United States)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  14. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    Science.gov (United States)

    Nolan, John P.; White, P. Scott

    2006-12-26

    Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

  15. Preparation and detection of nonradioactive nucleic acid and oligonucleotide

    International Nuclear Information System (INIS)

    There is increasing interest worldwide in the development of nucleic acid probes which are detected by nonradioactive means. In the research laboratory, the use of 32P for detection is undoubtedly the method of choice and is likely to remain so for the forseeable future, in spite of the half life of only 14 days for 32P. In the diagnostic laboratory on the other hand, the use of nonradioactive probes has many potential advantages. Perhaps the major one is that nonradioactive probes are stable for at least 6 to 12 months, and probably much longer if properly stored, thus leading to a substantial reduction in cost by obviating the need to prepare them every 2 to 3 weeks. In addition, there is no radiation exposure from routine daily use and there are no storage and disposal problems. Numerous methods are described in this chapter for the preparation by enzymatic and chemical techniques of nonradioactive nucleic acid and oligonucleotide probes. In many cases, the resulting probes have yet to be fully tested under hybridization conditions. In others, initial results look very promising since some nonradioactive probes can provide a sensitivity of detection of target sequences similar to that provided by 32P-labeled probes

  16. Radiation-induced electron migration in nucleic acids

    International Nuclear Information System (INIS)

    Radiation-induced electron migration along DNA is a mechanism by which randomly produced stochastic energy deposition events can lead to non-random types of damage along DNA manifested distal to the sites of the initial energy deposition. Radiation-induced electron migration in nucleic acids has been examined using oligonucleotides containing 5-bromouracil (5-BrU). Interaction of 5-BrU with solvated electrons results in release of bromide ions and formation of uracil-5-yl radicals. Monitoring either bromide ion release or uracil formation provides an opportunity to study electron migration processes in model nucleic acid systems. Using this approach we have discovered that electron migration along oligonucleotides is significantly influenced by the base sequence and strandedness. Migration along 7 base pairs in oligonucleotides containing guanine bases was observed for oligonucleotides irradiated in solution, which compares with mean migration distances of 6-10 bp for Escherichia coli DNA irradiated in solution and 5.5 bp for E. coli DNA irradiated in cells. Evidence also suggests that electron migration can occur preferentially in the 5' to 3' direction along a double-stranded oligonucleotide containing a region of purine bases adjacent to the 5-BrU moiety. Our continued efforts will provide information regarding the contribution of electron transfer along DNA to formation of locally multiply damaged sites created in DNA by exposure to ionizing radiation. (Author)

  17. 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.

  18. Extracellular Nucleic Acids in Blood of Patients with Chronic Obstructive Pulmonary Disease

    Directory of Open Access Journals (Sweden)

    Larissa E. Muravlyova

    2013-01-01

    Full Text Available The concentrations of extracellular nucleic acids and acid-soluble precursors of nucleic acids in blood of patients with different forms and severity of chronic obstructive pulmonary disease (COPD were evaluated. The significant increase of the content of extracellular RNA and acid-soluble precursors of nucleic acids in plasma of patients with COPD was detected. The decrease of extracellular RNA in plasma of patients with COPD worsening was diagnosed. Extracellular DNA in plasma and red blood cells of patients didn’t change significantly. The article examines the mechanisms of extracellular nucleic acids increase in blood of COPD patients, studies the possible role of extracellular RNA in development of coagulation disorders in COPD patients. The further research of the role of extracellular nucleic acids and their precursors in COPD progression is required

  19. Structural transformation induced by locked nucleic acid or 2′–O-methyl nucleic acid site-specific modifications on thrombin binding aptamer

    OpenAIRE

    Liu, Bo; Li, Da

    2014-01-01

    Background Locked nucleic acid (LNA) and 2'–O-methyl nucleic acid (OMeNA) are two of the most extensively studied nucleotide derivatives in the last decades. However, how they affect DNA quadruplex structures remains largely unknown. To explore their possible biological affinities for quadruplexes, we investigated how LNA- or OMeNA-substitutions affect G-quadruplex structure formation using a thrombin binding aptamer (TBA), the most studied extracorporal G-quadruplex-forming DNA sequence, whi...

  20. Apparatus for point-of-care detection of nucleic acid in a sample

    Energy Technology Data Exchange (ETDEWEB)

    Bearinger, Jane P.; Dugan, Lawrence C.

    2016-04-19

    Provided herein are methods and apparatus for detecting a target nucleic acid in a sample and related methods and apparatus for diagnosing a condition in an individual. The condition is associated with presence of nucleic acid produced by certain pathogens in the individual.

  1. 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...

  2. Comparative Assessment of Automated Nucleic Acid Sample Extraction Equipment for Biothreat Agents

    OpenAIRE

    Kalina, Warren Vincent; Douglas, Christina Elizabeth; Coyne, Susan Rajnik; Minogue, Timothy Devin

    2014-01-01

    Magnetic beads offer superior impurity removal and nucleic acid selection over older extraction methods. The performances of nucleic acid extraction of biothreat agents in blood or buffer by easyMAG, MagNA Pure, EZ1 Advanced XL, and Nordiag Arrow were evaluated. All instruments showed excellent performance in blood; however, the easyMAG had the best precision and versatility.

  3. Bis-Pyrene-Modified Unlocked Nucleic Acids: Synthesis, Hybridization Studies, and Fluorescent Properties

    Czech Academy of Sciences Publication Activity Database

    Perlíková, Pavla; Ejlersen, M.; Langkjaer, N.; Wengel, J.

    2014-01-01

    Roč. 9, č. 9 (2014), s. 2120-2127. ISSN 1860-7179 Grant ostatní: European Research Council(XE) FP7-268776 Institutional support: RVO:61388963 Keywords : fluorescence * nucleic acid hybridization * oligonucleotides * pyrenes * unlocked nucleic acids Subject RIV: CC - Organic Chemistry Impact factor: 2.968, year: 2014

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Science.gov (United States)

    2010-04-01

    ... assay. 866.3980 Section 866.3980 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended...

  6. [Determination of the nucleic acids in pig embryonic kidney cells by magnetic cytaphoresis].

    Science.gov (United States)

    Chikov, V M; Maksimova, E V

    1989-01-01

    Gallocyanine-chrome alum-stained pig embryonic kidney cells have paramagnetic properties. They move under the influence of gradient magnetic field (magnetophoresis). The velocity of magnetophoresis is proportional to the content of nucleic acids in cells. This allows to estimate the content of nucleic acids per cell dry weight by magnetophoresis and analytical centrifugation. PMID:2473104

  7. Quantifying 32P-labeled and unlabeled nucleic acids

    International Nuclear Information System (INIS)

    Recombinant DNA technology depends on detection methods for nucleic acids compatible with amounts ranging from picograms to grams and from tenths of a microliter to liters. In practical terms there are three basic techniques: (1) absorbance methods suitable for a minimum concentration of micrograms per milliliter, (2) fluorescence methods capable of detecting nanograms of DNA and micrograms of RNA, and (3) methods based on the detection of 32P. Because of the overwhelming importance in molecular biology of the third group, this chapter will stress exquisitely sensitive methods for measuring radioactivity in very small volumes. An illustration in which an enzyme-catalyzed reaction performed in 20 μl is monitored by consuming less than 2% of the total volume will be presented

  8. Spherical Nucleic Acids: A New Form of DNA

    Science.gov (United States)

    Cutler, Joshua Isaac

    Spherical Nucleic Acids (SNAs) are a new class of nucleic acid-based nanomaterials that exhibit unique properties currently being explored in the contexts of gene-based cancer therapies and in the design of programmable nanoparticle-based materials. The properties of SNAs differ from canonical, linear nucleic acids by virtue of their dense packing into an oriented 3-dimensional array. SNAs can be synthesized from a number of useful nanoparticle templates, such as plasmonic gold and silver, magnetic oxides, luminescent semi-conductor quantum dots, and silica. In addition, by crosslinking the oligonucleotides and dissolving the core, they can be made in a hollow form as well. This dissertation describes the evolution of SNAs from initial studies of inorganic nanoparticle-based materials densely functionalized with oligonucleotides to the proving of a hypothesis that their unique properties can be observed in a core-less structure if the nucleic acids are densely packed and highly oriented. Chapter two describes the synthesis of densely functionalized polyvalent oligonucleotide superparamagnetic iron oxide nanoparticles using the copper-catalyzed azide-alkyne cycloaddition reaction. These particles are shown to exhibit cooperative binding in a density- and salt concentration-dependent fashion, with nearly identical behaviors to those of SNA-functionalized gold nanoparticles. Importantly, these particles are the first non-gold particles shown to be capable of entering cells in high numbers via the SNA-mediated cellular uptake pathway, and provided the first evidence that SNA-mediated cellular uptake is core-independent. In the third chapter, a gold nanoparticle catalyzed alkyne cross-linking reaction is described that is capable of forming hollow organic nanoparticles using polymers with alkyne-functionalized backbones. With this method, the alkyne-modified polymers adsorb to the particle surfaces, cross-link on the surface, allowing the gold nanoparticle to be

  9. 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...

  10. Lipophilic nucleic acids--a flexible construction kit for organization and functionalization of surfaces.

    Science.gov (United States)

    Schade, Matthias; Berti, Debora; Huster, Daniel; Herrmann, Andreas; Arbuzova, Anna

    2014-06-01

    Lipophilic nucleic acids have become a versatile tool for structuring and functionalization of lipid bilayers and biological membranes as well as cargo vehicles to transport and deliver bioactive compounds, like interference RNA, into cells by taking advantage of reversible hybridization with complementary strands. This contribution reviews the different types of conjugates of lipophilic nucleic acids, and their physicochemical and self-assembly properties. Strategies for choosing a nucleic acid, lipophilic modification, and linker are discussed. Interaction with lipid membranes and its stability, dynamic structure and assembly of lipophilic nucleic acids upon embedding into biological membranes are specific points of the review. A large diversity of conjugates including lipophilic peptide nucleic acid and siRNA provides tailored solutions for specific applications in bio- and nanotechnology as well as in cell biology and medicine, as illustrated through some selected examples. PMID:24650567

  11. Current and future developments in nucleic acid-based diagnostics

    International Nuclear Information System (INIS)

    The detection and characterization of specific nucleic acids of medico-veterinary pathogens have proven invaluable for diagnostic purposes. Apart from hybridization and sequencing techniques, polymerase chain reaction (PCR) and numerous other methods have contributed significantly to this process. The integration of amplification and signal detection systems, including on-line real-time devices, have increased speed and sensitivity and greatly facilitated the quantification of target nucleic acids. They have also allowed for sequence characterization using melting or hybridization curves. Rugged portable real-time instruments for field use and robotic devices for processing samples are already available commercially. Various stem-loop DNA probes have been designed to have greater specificity for target recognition during real-time PCR. Various DNA fingerprinting techniques or post amplification sequencing are used to type pathogenic strains. Characterization according to DNA sequence is becoming more readily available as automated sequencers become more widely used. Reverse hybridization and to a greater degree DNA micro-arrays, are being used for genotyping related organisms and can allow for the detection of a large variety of different pathogens simultaneously. Non-radioactive labelling of DNA, especially using fluorophores and the principles of fluorescence resonance energy transfer, is now widely used, especially in real-time detection devices. Other detection methods include the use of surface plasmon resonance and MALDI-TOF mass spectrometry. In addition to these technological advances, contributions by bioinformatics and the description of unique signatures of DNA sequences from pathogens will contribute to the development of further assays for monitoring presence of pathogens. An important goal will be the development of robust devices capable of sensitively and specifically detecting a broad spectrum of pathogens that will be applicable for point

  12. 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.

  13. Shedding light on proteins, nucleic acids, cells, humans and fish

    Science.gov (United States)

    Setlow, Richard B.

    2002-01-01

    I was trained as a physicist in graduate school. Hence, when I decided to go into the field of biophysics, it was natural that I concentrated on the effects of light on relatively simple biological systems, such as proteins. The wavelengths absorbed by the amino acid subunits of proteins are in the ultraviolet (UV). The wavelengths that affect the biological activities, the action spectra, also are in the UV, but are not necessarily parallel to the absorption spectra. Understanding these differences led me to investigate the action spectra for affecting nucleic acids, and the effects of UV on viruses and cells. The latter studies led me to the discovery of the important molecular nature of the damages affecting DNA (cyclobutane pyrimidine dimers) and to the discovery of nucleotide excision repair. Individuals with the genetic disease xeroderma pigmentosum (XP) are extraordinarily sensitive to sunlight-induced skin cancer. The finding, by James Cleaver, that their skin cells were defective in DNA repair strongly suggested that DNA damage was a key step in carcinogenesis. Such information was important for estimating the wavelengths in sunlight responsible for human skin cancer and for predicting the effects of ozone depletion on the incidence of non-melanoma skin cancer. It took experiments with backcross hybrid fish to call attention to the probable role of the longer UV wavelengths not absorbed by DNA in the induction of melanoma. These reflections trace the biophysicist's path from molecules to melanoma.

  14. 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.

  15. Current and future developments in nucleic acid-based diagnostics

    International Nuclear Information System (INIS)

    The detection and characterization of specific nucleic acids of protozoa, rickettsia, bacteria and viruses have proven to be particularly useful for detecting pathogens of human and veterinary importance. It is also proving an invaluable tool for surveillance purposes and as a means of ensuring food security. Previous approaches towards pathogen isolation have often been tedious or even impossible. PCR, first conceived by Mullis in 1983, has proven to be a revolutionary technique for the rapid and accurate detection of numerous pathogens. The discovery and cloning of thermostable DNA polymerases has further contributed to this technology. Many additional developments, based on the basic principles of PCR, have been described e.g. RT-PCR, NASBA, RAPD, AFLP, LCR, PCR ELISA, strand displacement amplification (SDA), transcription-mediated amplification (TMA), branched DNA (bDNA), hybrid capture, immunocapture PCR. This list continues to expand with new variations on basic PCR principles. Improvements in thermocyclers involve the development of integrated amplification and signal detection systems, including on-line real-time devices. In addition, rugged portable instruments have been designed for field use. These are particularly useful as systems for early warning in detecting biowarfare agents and outbreaks of cross-boundary and other pathogens. Fluorophores, utilising principles of fluorescence resonance energy transfer, are used as labels for probes in such real-time assays. Molecular beacon technology also utilises such mechanisms. Real-time thermocyclers allow the monitoring of amplified DNA as well as establishing sequence characteristics based on melting or hybridisation curves. Taqman chemistry makes use of such a system. Stem-loop DNA probes have been designed to have increased specificity for target recognition and include molecular beacon methodologies, suppression PCR approaches and hairpin probes in DNA microarrays. Automated sample processing or robotic

  16. Multicenter evaluation of the Verigene Clostridium difficile nucleic acid assay.

    Science.gov (United States)

    Carroll, Karen C; Buchan, Blake W; Tan, Sokha; Stamper, Paul D; Riebe, Katherine M; Pancholi, Preeti; Kelly, Cheryl; Rao, Arundhati; Fader, Robert; Cavagnolo, Robert; Watson, Wendy; Goering, Richard V; Trevino, Ernest A; Weissfeld, Alice S; Ledeboer, Nathan A

    2013-12-01

    The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as "hypervirulent"; 53 were confirmed as ribotype

  17. Formulation of nucleic acid with pH-responsive amphipathic peptides for pulmonary delivery

    OpenAIRE

    Liang, Wanling; 梁婉玲

    2014-01-01

    Nucleic acids could be used as therapeutic agents for the treatment of many different diseases, but poor delivery limits their clinical application. A series of pH-responsive amphipathic peptides containing histidine or 2,3-diaminopropionic acid (Dap) derivatives, LAH and LADap peptides, were investigated in this study as nucleic acid carriers for the treatment of respiratory infectious disease. LAH and LADap peptides are cationic, amphipathic pH-responsive peptides. The major attractive ...

  18. Polymerase-directed synthesis of C5-ethynyl locked nucleic acids.

    Science.gov (United States)

    Veedu, Rakesh N; Burri, Harsha V; Kumar, Pawan; Sharma, Pawan K; Hrdlicka, Patrick J; Vester, Birte; Wengel, Jesper

    2010-11-15

    Modified nucleic acids have considerable potential in nanobiotechnology for the development of nanomedicines and new materials. Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far and we herein for the first time report the enzymatic incorporation of LNA-U and C5-ethynyl LNA-U nucleotides into oligonucleotides. Phusion High Fidelity and KOD DNA polymerases efficiently incorporated LNA-U and C5-ethynyl LNA-U nucleotides into a DNA strand and T7 RNA polymerase successfully accepted the LNA-U nucleoside 5'-triphosphate as substrate for RNA transcripts. PMID:20932755

  19. Recent developments in nucleic acid identification using solid-phase enzymatic assays

    International Nuclear Information System (INIS)

    This review covers recent achievements in the development of new approaches for enzymatically assisted detection of nucleic acids on microarrays. We discuss molecular techniques including the polymerase chain reaction, reverse transcription, allele specific primer extension and a range of isothermal techniques for the amplification and discrimination of nucleic acids. This also includes their implementation into microfluidic systems. These techniques all show great promise for use in the life sciences by allowing for high throughput, cost effective and highly sensitive and specific analysis of nucleic acids. Importantly, they can be potentially integrated into personalized and point-of-care medicine. (author)

  20. 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

    . Mass spectrometry (MS) has emerged as a sensitive and efficient analytical technique for determination of such cross-linking sites in proteins. The present review of the field describes a number of MS-based approaches for the characterization of cross-linked protein-nucleic acid complexes and for......Photochemical cross-linking is a commonly used method for studying the molecular details of protein-nucleic acid interactions. Photochemical cross-linking aids in defining nucleic acid binding sites of proteins via subsequent identification of cross-linked protein domains and amino acid residues...... 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...

  1. [Determination of body fluid based on analysis of nucleic acids].

    Science.gov (United States)

    Korabečná, Marie

    2015-01-01

    Recent methodological approaches of molecular genetics allow isolation of nucleic acids (DNA and RNA) from negligible forensic samples. Analysis of these molecules may be used not only for individual identification based on DNA profiling but also for the detection of origin of the body fluid which (alone or in mixture with other body fluids) forms the examined biological trace. Such an examination can contribute to the evaluation of procedural, technical and tactical value of the trace. Molecular genetic approaches discussed in the review offer new possibilities in comparison with traditional spectrum of chemical, immunological and spectroscopic tests especially with regard to the interpretation of mixtures of biological fluids and to the confirmatory character of the tests. Approaches based on reverse transcription of tissue specific mRNA and their subsequent polymerase chain reaction (PCR) and fragmentation analysis are applicable on samples containing minimal amounts of biological material. Methods for body fluid discrimination based on examination of microRNA in samples provided so far confusing results therefore further development in this field is needed. The examination of tissue specific methylation of nucleotides in selected gene sequences seems to represent a promising enrichment of the methodological spectrum. The detection of DNA sequences of tissue related bacteria has been established and it provides satisfactory results mainly in combination with above mentioned methodological approaches. PMID:26419517

  2. Crystal growth of proteins, nucleic acids, and viruses in gels.

    Science.gov (United States)

    Lorber, Bernard; Sauter, Claude; Théobald-Dietrich, Anne; Moreno, Abel; Schellenberger, Pascale; Robert, Marie-Claire; Capelle, Bernard; Sanglier, Sarah; Potier, Noëlle; Giegé, Richard

    2009-11-01

    Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses. PMID:20005247

  3. CNAPS VII - CIRCULATING NUCLEIC ACIDS IN PLASMA AND SERUM

    Directory of Open Access Journals (Sweden)

    Guest Editor: Damián García-Olmo, Spain

    2011-10-01

    Full Text Available On behalf of The 7th International Conference on Circulating Nucleic Acids in Plasma and Serum (CNAPS VII Organizing Committee, it is my great pleasure to extend a warm invitation to you to participate in the CNAPS VII, scheduled to be held the first time in Madrid, Spain, 24th and 25th October 2011, at the La Paz University Hospital. After the great success of the CNAPS VI at Hong Kong, we are honored with the opportunity to host the CNAPS VII in our city of  Madrid. To keep up with our bourgeoning field of science, an outstanding array of international experts on this field have agreed to share their latest results and experiences with us during this meeting, with elating debates, lectures and networking sessions. As in previous conferences, poster and oral presentation sessions will provide all participants with the opportunity to discuss their results with other experts in the field. Aside from the opportunities afforded by the conference´s working sessions, you will also have the chance to experience a myriad of Spanish tourist and cultural attractions. From classic visits to our famous museums, historical buildings and monuments, to a great variety of cultural activities and tours; from our delicious gastronomy to fabulous shopping bargains, there is plenty that will leave you with fond memories of Madrid and its warm and welcoming people.

  4. Nucleic Acid Nanostructures for Chemical and Biological Sensing.

    Science.gov (United States)

    Chandrasekaran, Arun Richard; Wady, Heitham; Subramanian, Hari K K

    2016-05-01

    The nanoscale features of DNA have made it a useful molecule for bottom-up construction of nanomaterials, for example, two- and three-dimensional lattices, nanomachines, and nanodevices. One of the emerging applications of such DNA-based nanostructures is in chemical and biological sensing, where they have proven to be cost-effective, sensitive and have shown promise as point-of-care diagnostic tools. DNA is an ideal molecule for sensing not only because of its specificity but also because it is robust and can function under a broad range of biologically relevant temperatures and conditions. DNA nanostructure-based sensors provide biocompatibility and highly specific detection based on the molecular recognition properties of DNA. They can be used for the detection of single nucleotide polymorphism and to sense pH both in solution and in cells. They have also been used to detect clinically relevant tumor biomarkers. In this review, recent advances in DNA-based biosensors for pH, nucleic acids, tumor biomarkers and cancer cell detection are introduced. Some challenges that lie ahead for such biosensors to effectively compete with established technologies are also discussed. PMID:27040036

  5. 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.

  6. Peptide nucleic acid (PNA) binding-mediated gene regulation

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Peptide nucleic acids (PNAs) are synthetic oligonucleotides with chemically modified backbones. PNAs can bind to both DNA and RNA targets in a sequence-specific manner to form PNA/DNA and PNA/RNA duplex structures. When bound to double-stranded DNA (dsDNA) targets, the PNA molecule replaces one DNA strand in the duplex by strand invasion to form a PNA/DNA/PNA [or (PNA)2/DNA] triplex structure and the displaced DNA strand exists as a singlestranded D-loop. PNA has been used in many studies as research tools for gene regulation and gene targeting. The Dloops generated from the PNA binding have also been demonstrated for its potential in initiating transcription and inducing gene expression. PNA provides a powerful tool to study the mechanism of transcription and an innovative strategy to regulate target gene expression. An understanding of the PNA-mediated gene regulation will have important clinical implications in treatment of many human diseases including genetic, cancerous, and age-related diseases.

  7. Quantum-Sequencing: Biophysics of quantum tunneling through nucleic acids

    Science.gov (United States)

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

    2014-03-01

    Tunneling microscopy and spectroscopy has extensively been used in physical surface sciences to study quantum tunneling to measure electronic local density of states of nanomaterials and to characterize adsorbed species. Quantum-Sequencing (Q-Seq) is a new method based on tunneling microscopy for electronic sequencing of single molecule of nucleic acids. A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free single-molecule sequencing method. Here, we present the unique ``electronic fingerprints'' for all nucleotides on DNA and RNA using Q-Seq along their intrinsic biophysical parameters. We have analyzed tunneling spectra for the nucleotides at different pH conditions and analyzed the HOMO, LUMO and energy gap for all of them. In addition we show a number of biophysical parameters to further characterize all nucleobases (electron and hole transition voltage and energy barriers). These results highlight the robustness of Q-Seq as a technique for next-generation sequencing.

  8. Linear and nonlinear optical properties of nucleic acid bases

    Science.gov (United States)

    Alparone, Andrea

    2013-01-01

    Electronic and vibrational (hyper)polarizabilities of neutral nucleic acid bases (uracil, thymine, cytosine, adenine, hypoxanthine and guanine) were determined using Hartree-Fock, correlated MPn (n = 2, 4), CCSD and DFT (B3LYP, B97-1, CAM-B3LYP) methods. The computations were performed in gaseous and aqueous phases for the most stable tautomeric forms. Frequency-dependent second-order hyperpolarizabilities were calculated for the OKE, IDRI, EFISHG and THG nonlinear optical processes at the wavelength of 1064 nm. The results show that the average electronic polarizabilities increase in the order uracil guanine. This order is also maintained for the electronic hyperpolarizabilities, with the inversion between cytosine and thymine. The response electric properties for the tautomers are almost similar to each other, whereas group substitution and solvation effects are much more significant. Among the DFT methods, the long-range corrected CAM-B3LYP functional gives the better performances, reproducing satisfactorily the correlated ab initio (hyper)polarizability data.

  9. [Development of Nucleic Acid-Based Adjuvant for Cancer Immunotherapy].

    Science.gov (United States)

    Kobiyama, Kouji; Ishii, Ken J

    2015-09-01

    Since the discovery of the human T cell-defined tumor antigen, the cancer immunotherapy field has rapidly progressed, with the research and development of cancer immunotherapy, including cancer vaccines, being conducted actively. However, the disadvantages of most cancer vaccines include relatively weak immunogenicity and immune escape or exhaustion. Adjuvants with innate immunostimulatory activities have been used to overcome these issues, and these agents have been shown to enhance the immunogenicity of cancer vaccines and to act as mono-therapeutic anti-tumor agents. CpG ODN, an agonist for TLR9, is one of the promising nucleic acid-based adjuvants, and it is a potent inducer of innate immune effector functions. CpG ODN suppresses tumor growth in the absence of tumor antigens and peptide administration. Therefore, CpG ODN is expected to be useful as a cancer vaccine adjuvant as well as a cancer immunotherapy agent. In this review, we discuss the potential therapeutic applications and mechanisms of CpG ODN for cancer immunotherapy. PMID:26469159

  10. Study on Suitability of KOD DNA Polymerase for Enzymatic Production of Artificial Nucleic Acids Using Base/Sugar Modified Nucleoside Triphosphates

    Directory of Open Access Journals (Sweden)

    Satoshi Obika

    2010-11-01

    Full Text Available Recently, KOD and its related DNA polymerases have been used for preparing various modified nucleic acids, including not only base-modified nucleic acids, but also sugar-modified ones, such as bridged/locked nucleic acid (BNA/LNA which would be promising candidates for nucleic acid drugs. However, thus far, reasons for the effectiveness of KOD DNA polymerase for such purposes have not been clearly elucidated. Therefore, using mutated KOD DNA polymerases, we studied here their catalytic properties upon enzymatic incorporation of nucleotide analogues with base/sugar modifications. Experimental data indicate that their characteristic kinetic properties enabled incorporation of various modified nucleotides. Among those KOD mutants, one achieved efficient successive incorporation of bridged nucleotides with a 2′-ONHCH2CH2-4′ linkage. In this study, the characteristic kinetic properties of KOD DNA polymerase for modified nucleoside triphosphates were shown, and the effectiveness of genetic engineering in improvement of the enzyme for modified nucleotide polymerization has been demonstrated.

  11. A conversational system for the computer analysis of nucleic acid sequences.

    OpenAIRE

    Sege, R; Söll, D.; Ruddle, F H; Queen, C

    1981-01-01

    We present a conversational system for the computer analysis of nucleic acid and protein sequences based on the well-known Queen and Korn program (1). The system can be used by persons with only minimal knowledge of computers.

  12. 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...

  13. Peptide nucleic acid: a new artificial biomacromolecular with great potential applications in molecular biology and biomedicine

    Institute of Scientific and Technical Information of China (English)

    WANG Jin-ke; LU Zu-hong; HE Nong-yue

    2001-01-01

    @@ Peptide nucleic acid (PNA) is a DNA mimic that was originally developed by Peter E Nielsen in 1991 as a reagent for sequence-specific recognition of double stranded (ds) DNA by a conventional triple helix type principle.

  14. 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...

  15. Novel redox-sensing modules : Accessory protein- and nucleic acid-mediated signaling

    NARCIS (Netherlands)

    Siedenburg, Gabriele; Groves, Matthew R; Ortiz de Orué Lucana, Darío

    2012-01-01

    SIGNIFICANCE: Organisms have evolved both enzymatic and nonenzymatic pathways to prevent oxidative damage to essential macromolecules, including proteins and nucleic acids. Pathways modulated by different protein-based sensory and regulatory modules ensure a rapid and appropriate response. RECENT AD

  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 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. PMID:26482088

  18. In-silico design of computational nucleic acids for molecular information processing.

    Science.gov (United States)

    Ramlan, Effirul Ikhwan; Zauner, Klaus-Peter

    2013-01-01

    Within recent years nucleic acids have become a focus of interest for prototype implementations of molecular computing concepts. During the same period the importance of ribonucleic acids as components of the regulatory networks within living cells has increasingly been revealed. Molecular computers are attractive due to their ability to function within a biological system; an application area extraneous to the present information technology paradigm. The existence of natural information processing architectures (predominately exemplified by protein) demonstrates that computing based on physical substrates that are radically different from silicon is feasible. Two key principles underlie molecular level information processing in organisms: conformational dynamics of macromolecules and self-assembly of macromolecules. Nucleic acids support both principles, and moreover computational design of these molecules is practicable. This study demonstrates the simplicity with which one can construct a set of nucleic acid computing units using a new computational protocol. With the new protocol, diverse classes of nucleic acids imitating the complete set of boolean logical operators were constructed. These nucleic acid classes display favourable thermodynamic properties and are significantly similar to the approximation of successful candidates implemented in the laboratory. This new protocol would enable the construction of a network of interconnecting nucleic acids (as a circuit) for molecular information processing. PMID:23647621

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

    OpenAIRE

    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

    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...

  20. Theoretical modeling of the metal ion effects on NMR parameters in nucleic acid backbone

    Czech Academy of Sciences Publication Activity Database

    Benda, Ladislav; Schneider, Bohdan; Sychrovský, Vladimír

    Florence: -, 2010. s. 420-420. [WWMR2010. Joint EUROMAR 2010 and ISMAR Conference /17./. 04.07.2010-09.07.2010, Florence] R&D Projects: GA ČR GAP205/10/0228 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520701 Keywords : nucleic acid phosphate * nucleic acids solvation * magnesium ion Subject RIV: CF - Physical ; Theoretical Chemistry

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

    OpenAIRE

    Matos, T.; Senkbeil, Silja; Mendonça, A; Queiroz, J. A.; Kutter, Jörg Peter; Bulow, L.

    2013-01-01

    Due to the extensive use of nucleic acid and protein analysis of bacterial samples, there is a need for simple and rapid extraction protocols for both plasmid DNA and RNA molecules as well as reporter proteins like the green fluorescent protein (GFP). In this report, an electropermeability 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 microflu...

  2. An instrument for automated purification of nucleic acids from contaminated forensic samples

    OpenAIRE

    Broemeling, David J; Pel, Joel; Gunn, Dylan C; Mai, Laura; Thompson, Jason D.; Poon, Hiron; Marziali, Andre

    2008-01-01

    Forensic crime scene sample analysis, by its nature, often deals with samples in which there are low amounts of nucleic acids, on substrates that often lead to inhibition of subsequent enzymatic reactions such as PCR amplification for STR profiling. Common substrates include denim from blue jeans, which yields indigo dye as a PCR inhibitor, and soil, which yields humic substances as inhibitors. These inhibitors frequently co-extract with nucleic acids in standard column or bead-based preps, l...

  3. Detergent-enabled transport of proteins and nucleic acids through hydrophobic solvents.

    OpenAIRE

    Bromberg, L E; Klibanov, A M

    1994-01-01

    It is demonstrated that proteins and nucleic acids can be transported through hydrophobic organic solvents (liquid membranes) via nonspecific complex formation with detergents, whereas no macromolecule transport is observed without the latter. A protein (or a nucleic acid) first interacts with an oppositely charged detergent due to hydrophobic ion pairing in the aqueous feed phase. The resultant hydrophobic complex readily partitions into an organic solvent and then into the aqueous receiver ...

  4. 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. PMID:19507821

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

    Directory of Open Access Journals (Sweden)

    Dominique Lévesque

    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.

  6. Intracellular fate of spherical nucleic acid nanoparticle conjugates.

    Science.gov (United States)

    Wu, Xiaochen A; Choi, Chung Hang J; Zhang, Chuan; Hao, Liangliang; Mirkin, Chad A

    2014-05-28

    Spherical nucleic acid (SNA) nanoparticle conjugates are a class of bionanomaterials that are extremely potent in many biomedical applications. Their unique ability to enter multiple mammalian cell types as single-entity agents arises from their novel three-dimensional architecture, which consists of a dense shell of highly oriented oligonucleotides chemically attached typically to a gold nanoparticle core. This architecture allows SNAs to engage certain cell surface receptors to facilitate entry. Here, we report studies aimed at determining the intracellular fate of SNAs and the trafficking events that occur inside C166 mouse endothelial cells after cellular entry. We show that SNAs traffic through the endocytic pathway into late endosomes and reside there for up to 24 h after incubation. Disassembly of oligonucleotides from the nanoparticle core is observed 16 h after cellular entry, most likely due to degradation by enzymes such as DNase II localized in late endosomes. Our observations point to these events being likely independent of core composition and treatment conditions, and they do not seem to be particularly dependent upon oligonucleotide sequence. Significantly and surprisingly, the SNAs do not enter the lysosomes under the conditions studied. To independently track the fate of the particle core and the fluorophore-labeled oligonucleotides that comprise its shell, we synthesized a novel class of quantum dot SNAs to determine that as the SNA structures are broken down over the 24 h time course of the experiment, the oligonucleotide fragments are recycled out of the cell while the nanoparticle core is not. This mechanistic insight points to the importance of designing and synthesizing next-generation SNAs that can bypass the degradation bottleneck imposed by their residency in late endosomes, and it also suggests that such structures might be extremely useful for endosomal signaling pathways by engaging receptors that are localized within the endosome

  7. Noninvasive nucleic acid-based approaches to monitor placental health and predict pregnancy-related complications.

    Science.gov (United States)

    Manokhina, Irina; Wilson, Samantha L; Robinson, Wendy P

    2015-10-01

    During pregnancy, the placenta releases a variety of nucleic acids (including deoxyribonucleic acid, messenger ribonucleic acid, or microribonucleic acids) either as a result of cell turnover or as an active messaging system between the placenta and cells in the maternal body. The profile of released nucleic acids changes with the gestational age and has been associated with maternal and fetal parameters. It also can directly reflect pathological changes in the placenta. Nucleic acids may therefore provide a rich source of novel biomarkers for the prediction of pregnancy complications. However, their utility in the clinical setting depends, first, on overcoming some technical considerations in their quantification, and, second, on developing a better understanding of the factors that influence their function and abundance. PMID:26428499

  8. 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.

  9. Multiplexed analysis of genes using nucleic acid-stabilized silver-nanocluster quantum dots.

    Science.gov (United States)

    Enkin, Natalie; Wang, Fuan; Sharon, Etery; Albada, H Bauke; Willner, Itamar

    2014-11-25

    Luminescent nucleic acid-stabilized Ag nanoclusters (Ag NCs) are applied for the optical detection of DNA and for the multiplexed analysis of genes. Two different sensing modules including Ag NCs as luminescence labels are described. One sensing module involves the assembly of a three-component sensing module composed of a nucleic acid-stabilized Ag NC and a quencher-modified nucleic acid hybridized with a nucleic acid scaffold that is complementary to the target DNA. The luminescence of the Ag NCs is quenched in the sensing module nanostructure. The strand displacement of the scaffold by the target DNA separates the nucleic acid-functionalized Ag NCs, leading to the turned-on luminescence of the NCs and to the optical readout of the sensing process. By implementing two different-sized Ag NC-modified sensing modules, the parallel multiplexed analysis of two genes (the Werner Syndrome gene and the HIV, human immunodeficiency, gene), using 615 and 560 nm luminescent Ag NCs, is demonstrated. The second sensing module includes the nucleic acid functionalized Ag NCs and the quencher-modified nucleic acid hybridized with a hairpin DNA scaffold. The luminescence of the Ag NCs is quenched in the sensing module. Opening of the hairpin by the target DNA triggers the luminescence of the Ag NCs, due to the spatial separation of the Ag NCs/quencher units. The system is applied for the optical detection of the BRAC1 gene. In addition, by implementing two-sized Ag NCs, the multiplexed analysis of two genes by the hairpin sensing module approach is demonstrated. PMID:25327411

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

    DEFF Research Database (Denmark)

    Wittung, P; Nielsen, Peter E.; Buchardt, O; Egholm, M; Nordén, B

    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...... of double helical DNA-like structures in solution....

  11. Complex formation of cadmium with sugar residues, nucleobases, phosphates, nucleotides, and nucleic acids.

    Science.gov (United States)

    Sigel, Roland K O; Skilandat, Miriam; Sigel, Astrid; Operschall, Bert P; Sigel, Helmut

    2013-01-01

    Cadmium(II), commonly classified as a relatively soft metal ion, prefers indeed aromatic-nitrogen sites (e.g., N7 of purines) over oxygen sites (like sugar-hydroxyl groups). However, matters are not that simple, though it is true that the affinity of Cd(2+) towards ribose-hydroxyl groups is very small; yet, a correct orientation brought about by a suitable primary binding site and a reduced solvent polarity, as it is expected to occur in a folded nucleic acid, may facilitate metal ion-hydroxyl group binding very effectively. Cd(2+) prefers the guanine(N7) over the adenine(N7), mainly because of the steric hindrance of the (C6)NH(2) group in the adenine residue. This Cd(2+)-(N7) interaction in a guanine moiety leads to a significant acidification of the (N1)H meaning that the deprotonation reaction occurs now in the physiological pH range. N3 of the cytosine residue, together with the neighboring (C2)O, is also a remarkable Cd(2+) binding site, though replacement of (C2)O by (C2)S enhances the affinity towards Cd(2+) dramatically, giving in addition rise to the deprotonation of the (C4)NH(2) group. The phosphodiester bridge is only a weak binding site but the affinity increases further from the mono- to the di- and the triphosphate. The same also holds for the corresponding nucleotides. Complex stability of the pyrimidine-nucleotides is solely determined by the coordination tendency of the phosphate group(s), whereas in the case of purine-nucleotides macrochelate formation takes place by the interaction of the phosphate-coordinated Cd(2+) with N7. The extents of the formation degrees of these chelates are summarized and the effect of a non-bridging sulfur atom in a thiophosphate group (versus a normal phosphate group) is considered. Mixed ligand complexes containing a nucleotide and a further mono- or bidentate ligand are covered and it is concluded that in these species N7 is released from the coordination sphere of Cd(2+). In the case that the other ligand

  12. Screen printing of nucleic acid detecting carbon electrodes.

    Science.gov (United States)

    Dequaire, Murielle; Heller, Adam

    2002-09-01

    A large fraction of the presently mass-manufactured (> 10(8) units/year) electrochemical biosensors, used mostly by diabetic people to monitor their blood glucose levels, have screen-printed carbon working electrodes. An earlier study (Campbell, C. N., et al. Anal. Chem. 2002, 74, 158-162) showed that nucleic acids can be assayed at 1 nM concentrations by a sandwich-type amperometric method. The assay was performed with vitreous carbon working electrodes on which an electron-conducting polycationic redox polymer and avidin were coelectrodeposited. Because the rate of the electrodeposition increases with the surface density of the polycationic redox polymer, its practicality depends on pretreatment of the surface, which adds anionic functions. (Gao, Z., et al. Angew. Chem. Int. Ed. 2002, 41, 810-813). Here it is shown that the required conducting redox polymer films can be electrodeposited on potentially mass manufacturable electrodes made by screen-printing hydrophilic carbon inks on polyester sheets. The modified electrodes are made in two steps. First a polycationic electron-conducting redox polymer is cross-linked and electrodeposited by applying a negative potential. Next, an amine-terminated 20-base single-stranded oligonucleotide is electrodeposited by ligand-exchange. Both steps involve exchange of a labile inner sphere chloride ligand of the polymer-bound osmium-complex: Cross-linking and electrodeposition of the redox polymer result when inner-sphere chloride anions of the osmium complexes are exchanged by imidazole functions of neighboring chains. Incorporation of the oligonucleotide in the redox polymer results in the formation of a coordinative bond between the terminal amine (attached through a spacer to the oligonucleotide) and the osmium complex. In testing for the presence of a 38-base oligonucleotide, the analyte, in a 15- or 25-microL droplet of hybridization solution, is hybridized with and captured by the 20-base electrode-bound sequence; then

  13. Low ionizing radiation influence on the nucleic acids in common Acacia

    International Nuclear Information System (INIS)

    Three months old common acacia saplings were selected from a forestry nursery with an uniform genetic background. A 60 Co ionizing radiation source, with a dose rate of 10 mCi was used to irradiate saplings for different time durations: 1h, 2h, 4h and 7 h. One day after the irradiation, small amounts of green tissue were took for nucleic acid extraction and assay. The quantitative extraction was performed in perchloric acid at a temperature of about 100 Celsius degrees. Centrifugation at 5,000 cycles/minute was performed and supernatant liquid was used for spectrophotometric assay. The light extinction at the wavelengths of 270 nm and 290 nm was measured, after Spirin's method using a Beckman spectrophotometer. The average between DNA and RNA content values was evaluated and represented graphically. The logarithmic representation of nucleic acids content, via exposure time, fitted with a mathematical polynomial function of second order, showed the decreasing of nucleic acids content in samples in comparison to the control. This could be the effect of radiation damage at the level of the nucleic acids primary structure, resulting in the reducing of nucleic acid amount in the vegetal cells of irradiated common acacia saplings. Direct radiation action on some chemical bonds as well as indirect effects mediated by water radiolysis could be implied in the diminution of DNA and RNA content from common acacia cell nuclei. (authors)

  14. Multivalent ion-mediated nucleic acid helix-helix interactions: RNA versus DNA

    CERN Document Server

    Wu, Yuan-Yan; Zhang, Jin-Si; Zhu, Xiao-Long; Tan, Zhi-Jie

    2015-01-01

    Ion-mediated interaction is critical to the structure and stability of nucleic acids. Recent experiments suggest that the multivalent ion-induced aggregation of double-stranded (ds) RNAs and DNAs may strongly depend on the topological nature of helices, while there is still lack of an understanding on the relevant ion-mediated interactions at atomistic level. In this work, we have directly calculated the potentials of mean force (PMF) between two dsRNAs and between two dsDNAs in Cobalt Hexammine ion (Co-Hex) solutions by the atomistic molecular dynamics simulations. Our calculations show that at low [Co-Hex], the PMFs between B-DNAs and between A-RNAs are both (strongly) repulsive.However, at high [Co-Hex], the PMF between B-DNAs is strongly attractive, while those between A-RNAs and between A-DNAs are still (weakly) repulsive. The microscopic analyses show that for A-form helices, Co-Hex would become internal binding into the deep major groove and consequently cannot form the evident ion-bridge between adjac...

  15. Use of Chromophoric Ligands to Visually Screen Co-crystals of Putative Protein-Nucleic Acid Complexes

    OpenAIRE

    Jiang, Xiaohua; Egli, Martin

    2011-01-01

    Distinguishing between crystals of protein-nucleic acid complexes and those containing protein alone is a common problem in structural studies of protein-nucleic acid interactions. Currently there are several methods available for detecting nucleic acid in crystals, including gel electrophoresis, SYBR Gold fluorescence dye staining and methyl violet staining. However, they require either that the crystals be sacrificed or access to a fluorescence microscope. In this protocol, we describe an a...

  16. Quantitative and discriminative analysis of nucleic acid samples using luminometric nonspecific nanoparticle methods

    Science.gov (United States)

    Pihlasalo, S.; Mariani, L.; Härmä, H.

    2016-03-01

    Homogeneous simple assays utilizing luminescence quenching and time-resolved luminescence resonance energy transfer (TR-LRET) were developed for the quantification of nucleic acids without sequence information. Nucleic acids prevent the adsorption of a protein to europium nanoparticles which is detected as a luminescence quenching of europium nanoparticles with a soluble quencher or as a decrease of TR-LRET from europium nanoparticles to the acceptor dye. Contrary to the existing methods based on fluorescent dye binding to nucleic acids, equal sensitivities for both single- (ssDNA) and double-stranded DNA (dsDNA) were measured and a detection limit of 60 pg was calculated for the quenching assay. The average coefficient of variation was 5% for the quenching assay and 8% for the TR-LRET assay. The TR-LRET assay was also combined with a nucleic acid dye selective to dsDNA in a single tube assay to measure the total concentration of DNA and the ratio of ssDNA and dsDNA in the mixture. To our knowledge, such a multiplexed assay is not accomplished with commercially available assays.Homogeneous simple assays utilizing luminescence quenching and time-resolved luminescence resonance energy transfer (TR-LRET) were developed for the quantification of nucleic acids without sequence information. Nucleic acids prevent the adsorption of a protein to europium nanoparticles which is detected as a luminescence quenching of europium nanoparticles with a soluble quencher or as a decrease of TR-LRET from europium nanoparticles to the acceptor dye. Contrary to the existing methods based on fluorescent dye binding to nucleic acids, equal sensitivities for both single- (ssDNA) and double-stranded DNA (dsDNA) were measured and a detection limit of 60 pg was calculated for the quenching assay. The average coefficient of variation was 5% for the quenching assay and 8% for the TR-LRET assay. The TR-LRET assay was also combined with a nucleic acid dye selective to dsDNA in a single tube

  17. Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine.

    Science.gov (United States)

    Egatz-Gomez, Ana; Wang, Ceming; Klacsmann, Flora; Pan, Zehao; Marczak, Steve; Wang, Yunshan; Sun, Gongchen; Senapati, Satyajyoti; Chang, Hsueh-Chia

    2016-05-01

    Nucleic acid biomarkers have enormous potential in non-invasive diagnostics and disease management. In medical research and in the near future in the clinics, there is a great demand for accurate miRNA, mRNA, and ctDNA identification and profiling. They may lead to screening of early stage cancer that is not detectable by tissue biopsy or imaging. Moreover, because their cost is low and they are non-invasive, they can become a regular screening test during annual checkups or allow a dynamic treatment program that adjusts its drug and dosage frequently. We briefly review a few existing viral and endogenous RNA assays that have been approved by the Federal Drug Administration. These tests are based on the main nucleic acid detection technologies, namely, quantitative reverse transcription polymerase chain reaction (PCR), microarrays, and next-generation sequencing. Several of the challenges that these three technologies still face regarding the quantitative measurement of a panel of nucleic acids are outlined. Finally, we review a cluster of microfluidic technologies from our group with potential for point-of-care nucleic acid quantification without nucleic acid amplification, designed to overcome specific limitations of current technologies. We suggest that integration of these technologies in a modular design can offer a low-cost, robust, and yet sensitive/selective platform for a variety of precision medicine applications. PMID:27190565

  18. Lipid-Nucleic Acid Supramolecular Complexes: Lipoplex Structure and the Kinetics of Formation

    Directory of Open Access Journals (Sweden)

    Nily Dan

    2015-06-01

    Full Text Available The need for synthetic gene therapy or gene silencing vehicles that can insert therapeutic nucleic acids (DNA or siRNA into cells (so-called transfection has focused interest on lipid-nucleic acid assemblies (lipoplexes. This paper reviews the kinetics pathways leading to lipoplex formation and structure. The process is qualitatively comparable to those of cluster nucleation and growth and to the adsorption of polyelectrolytes on colloidal particles: Initially is a rapid stage where the nucleic acid binds onto the surface of the cationic lipid aggregate (adsorption, or nucleation. This is followed by an intermediate step where the lipid/nucleic acid complexes flocculate to form larger structures (growth. The last and final step involves internal rearrangement, where the overall global structure remains constant while local adjustment of the nucleic acid/lipid organization takes place until the equilibrium lipoplex characteristics are obtained. This step can require unusually long time scales of order hours or longer. Understanding the kinetics of lipoplex formation is not only of fundamental interest as a multi-component, multi-length scale and multi-time scale process, but also has significant implications for the utilization of lipoplexes as carriers for gene delivery and gene silencing agents.

  19. Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays.

    Science.gov (United States)

    Lambert, Amy J; Martin, Denise A; Lanciotti, Robert S

    2003-01-01

    We have developed nucleic acid sequence-based amplification (NASBA), standard reverse transcription PCR (RT-PCR), and TaqMan nucleic acid amplification assays for the rapid detection of North American eastern equine encephalitis (EEE) and western equine encephalitis (WEE) viral RNAs from samples collected in the field and clinical samples. The sensitivities of these assays have been compared to that of virus isolation. While all three types of nucleic acid amplification assays provide rapid detection of viral RNAs comparable to the isolation of viruses in Vero cells, the TaqMan assays for North American EEE and WEE viral RNAs are the most sensitive. We have shown these assays to be specific for North American EEE and WEE viral RNAs by testing geographically and temporally distinct strains of EEE and WEE viruses along with a battery of related and unrelated arthropodborne viruses. In addition, all three types of nucleic acid amplification assays have been used to detect North American EEE and WEE viral RNAs from mosquito and vertebrate tissue samples. The sensitivity, specificity, and rapidity of nucleic acid amplification demonstrate the usefulness of NASBA, standard RT-PCR, and TaqMan assays, in both research and diagnostic settings, to detect North American EEE and WEE viral RNAs. PMID:12517876

  20. Concentration methods for high-resolution THz spectroscopy of nucleic-acid biomolecules and crystals

    Science.gov (United States)

    Brown, E. R.; Zhang, W.; Mendoza, E. A.; Kuznetsova, Y.; Brueck, S. R. J.; Rahman, M.; Norton, M. L.

    2012-03-01

    Biomolecules can exhibit low-lying vibrational modes in the THz region which are detectable in transmission given a strong molecular dipole moment and optical depth, and a spectrometer of adequate sensitivity. The nucleic acids are particularly interesting because of applications such as label-free gene assay, bio-agent detection, etc. However for nucleic acids, sample preparation and THz coupling are of paramount importance because of the strong absorption by liquid water and the small concentration of molecules present in physiological solutions. Concentration methods become necessary to make the THz vibrational modes detectable, either by concentrating the nucleic-acid sample itself in a small volume but large area, or by concentrating the THz radiation down to the volume of the sample. This paper summarizes one type of the first method: nanofluidic channel arrays for biological nucleic acids; and two types of the second method: (1) a circular-waveguide pinhole, and (2) a circular-waveguide, conical-horn coupling structure, both for DNA crystals. The first method has been demonstrated on a very short artificial nucleic acid [small-interfering (si) RNA (17-to-25 bp)] and a much longer, biological molecule [Lambda-phage DNA (48.5 kbp)]. The second method has been demonstrated on small (~100 micron) single crystals of DNA grown by the sitting-drop method.

  1. 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

    2016-02-01

    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. PMID:26717419

  2. 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.

  3. High-resolution, hybrid optical trapping methods, and their application to nucleic acid processing proteins.

    Science.gov (United States)

    Chemla, Yann R

    2016-10-01

    Optical tweezers have become a powerful tool to investigate nucleic-acid processing proteins at the single-molecule level. Recent advances in this technique have now enabled measurements resolving the smallest units of molecular motion, on the scale of a single base pair of DNA. In parallel, new instrumentation combining optical traps with other functionalities have been developed, incorporating mechanical manipulation along orthogonal directions or fluorescence imaging capabilities. Here, we review these technical advances, their capabilities, and limitations, focusing on benchmark studies of protein-nucleic acid interactions they have enabled. We highlight recent work that combines several of these advances together and its application to nucleic-acid processing enzymes. Finally, we discuss future prospects for these exciting developments. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 704-714, 2016. PMID:27225537

  4. MATra - Magnet Assisted Transfection: combining nanotechnology and magnetic forces to improve intracellular delivery of nucleic acids.

    Science.gov (United States)

    Bertram, J

    2006-08-01

    Recent efforts combining nanotechnology and magnetic properties resulted in the development and commercialization of magnetic nanoparticles that can be used as carriers for nucleic acids for in vitro transfection and for gene therapy approaches including DNA-based vaccination strategies. The efficiency of intracellular delivery is still a limiting factor for basic cell biological research and also for emerging technologies such as temporary gene silencing based on inhibitory RNA/siRNA. Nanotechnology has resulted in a variety of different nanostructures and especially nanoparticles as carriers in a wide range of new drug delivery systems for conventional drugs, recombinant proteins, vaccines and more recently nucleic acids. It is possible to combine superparamagnetic nanoparticles with magnetic forces to increase, direct and optimize intracellular delivery of biomolecules. This article discusses the main approaches in the field of magnet assisted transfection (MATra) focusing on the transfection or intracellular delivery of nucleic acids, although also suitable to improve the intracellular delivery of other biomolecules. PMID:16918404

  5. Enhancing aptamer function and stability via in vitro selection using modified nucleic acids.

    Science.gov (United States)

    Meek, Kirsten N; Rangel, Alexandra E; Heemstra, Jennifer M

    2016-08-15

    Nucleic acid aptamers have emerged as a promising alternative to antibodies for use as recognition elements in therapeutics, bioimaging, and analytical applications. A key benefit that aptamers possess relative to antibodies is their ability to be chemically synthesized. This advantage, coupled with the broad range of modified nucleotide building blocks that can be constructed using chemical synthesis, has enabled the discovery and development of modified aptamers having extraordinary affinity, specificity, and biostability. Early efforts to generate modified aptamers focused on selection of a native DNA or RNA aptamer, followed by post-selection trial-and-error testing of modifications. However, recent advances in polymerase engineering and templated nucleic acid synthesis have enabled the direct selection of aptamers having modified backbones and nucleobases. This review will discuss these technological advances and highlight the improvements in aptamer function that have been realized through in vitro selection of non-natural nucleic acids. PMID:27012179

  6. Labelling of nucleic acid components with tritium by hydrogenolysis of corresponding precursors

    International Nuclear Information System (INIS)

    To desalt the luates of liquid column chromatography containing components of the nucleic acids different types of activated carbons are used: AG-5, Ou-4, KAD, BAU (SU), Norit (GB) and Carboafin (CS). The Carborafin (CS) carbon proved to be the most efficient for the purpose. Dependences of the adsorption degree on pH, the time of the phases contact, temperature, concentration of the salt background (ammonium formite, lithium chloride) as well as adsorption isotherm are determined for the activated carbon. Desorption conditions of the nucleic acids components from the carbon are studied. It is shown that quantitative desorption is achieved when 1n solution of ammonia is used in 50% ethanole for 50-60 min. Data on practical application of the method to desalt the eluates containing tritiated nucleic acid components with a high activity are presented

  7. A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification

    Directory of Open Access Journals (Sweden)

    Pascal Craw

    2015-09-01

    Full Text Available Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings.

  8. Theoretical study of the influence of ribose on the proton transfer phenomenon of nucleic acid bases

    International Nuclear Information System (INIS)

    The first comprehensive theoretical study of ribose's effects on the behavior of proton transfer of nucleic acid base is presented. The specific hydrogen bonding of the ribose hydroxyls plays a very important role in the stabilization of the structure of ribonucleoside. Nine stable uridine conformations have been reported. The intermolecular proton transfer of the isolated, monohydrated uridine complexes in three different regions were extensively explored on the basis of density functional theory at the B3LYP/6-31+G* level. With the introduction of the ribose, not only the structural parameters of the nucleic acid bases changed, but also the energy barriers of the proton transfer process changed. Furthermore, changes of the electron distributions of the molecular orbital of the nucleic acid bases were also analyzed by NBO analysis. Consideration of the ribose's influence represents a much more real situation in the RNA

  9. Structure and behaviour of proteins, nucleic acids and viruses from vibrational Raman optical activity

    DEFF Research Database (Denmark)

    Barron, L.D.; Blanch, E.W.; McColl, I.H.; Syme, C.D.; Hecht, L.; Nielsen, Kurt

    aqueous solution. Protein ROA spectra provide information on the secondary and tertiary structure of the polypeptide backbone, hydration, side chain conformation and structural elements present in denatured states. Nucleic acid ROA spectra provide information on the sugar ring conformation, the base...... stacking arrangement and the mutual orientation of the sugar and base rings around the C-N glycosidic link. The ROA spectra of intact viruses provide information on the folds of the coat proteins and the nucleic acid structure. The large number of structure-sensitive bands in protein ROA spectra is...... especially favourable for fold determination using pattern recognition techniques. This article gives a brief account of the ROA technique and presents the ROA spectra of a selection of proteins, nucleic acids and viruses that illustrate the applications of ROA spectroscopy in biomolecular research....

  10. 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. PMID:19722647

  11. Salt Contribution to the Flexibility of Single-stranded Nucleic Acid of Finite Length

    CERN Document Server

    Wang, Feng-Hua; Tan, Zhi-Jie

    2012-01-01

    Nucleic acids are negatively charged macromolecules and their structure properties are strongly coupled to metal ions in solutions. In this paper, the salt effects on the flexibility of single stranded (ss) nucleic acid chain ranging from 12 to 120 nucleotides are investigated systematically by the coarse grained Monte Carlo simulations where the salt ions are considered explicitly and the ss chain is modeled with the virtual bond structural model. Our calculations show that, the increase of ion concentration causes the structural collapse of ss chain and multivalent ions are much more efficient in causing such collapse, and trivalent and small divalent ions can both induce more compact state than a random relaxation state. We found that monovalent, divalent and trivalent ions can all overcharge ss chain, and the dominating source for such overcharging changes from ion exclusion volume effect to ion Coulomb correlations. In addition, the predicted Na and Mg dependent persistence length lp of ss nucleic acid a...

  12. From keys to bulldozers: expanding roles for winged helix domains in nucleic-acid-binding proteins.

    Science.gov (United States)

    Harami, Gábor M; Gyimesi, Máté; Kovács, Mihály

    2013-07-01

    The winged helix domain (WHD) is a widespread nucleic-acid-binding protein structural element found in all kingdoms of life. Although the overall structure of the WHD is conserved, its functional properties and interaction profiles are extremely versatile. WHD-containing proteins can exploit nearly the full spectrum of nucleic acid structural features for recognition and even covalent modification or noncovalent rearrangement of target molecules. WHD functions range from sequence-recognizing keys in transcription factors and bulldozer-like strand-separating wedges in helicases to mediators of protein-protein interactions (PPIs). Further investigations are needed to understand the contribution of WHD structural dynamics to nucleic-acid-modifying enzymatic functions. PMID:23768997

  13. A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification.

    Science.gov (United States)

    Craw, Pascal; Mackay, Ruth E; Naveenathayalan, Angel; Hudson, Chris; Branavan, Manoharanehru; Sadiq, S Tariq; Balachandran, Wamadeva

    2015-01-01

    Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings. PMID:26389913

  14. Methods of staining target chromosomal DNA employing high complexity nucleic acid probes

    Science.gov (United States)

    Gray, Joe W.; Pinkel, Daniel; Kallioniemi, Ol'li-Pekka; Kallioniemi, Anne; Sakamoto, Masaru

    2006-10-03

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML), retinoblastoma, ovarian and uterine cancers, and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

  15. The in Silico Insight into Carbon Nanotube and Nucleic Acid Bases Interaction

    Science.gov (United States)

    Karimi, Ali Asghar; Ghalandari, Behafarid; Tabatabaie, Seyed Saleh; Farhadi, Mohammad

    2016-01-01

    Background To explore practical applications of carbon nanotubes (CNTs) in biomedical fields the properties of their interaction with biomolecules must be revealed. Recent years, the interaction of CNTs with biomolecules is a subject of research interest for practical applications so that previous research explored that CNTs have complementary structure properties with single strand DNA (ssDNA). Objectives Hence, the quantum mechanics (QM) method based on ab initio was used for this purpose. Therefore values of binding energy, charge distribution, electronic energy and other physical properties of interaction were studied for interaction of nucleic acid bases and SCNT. Materials and Methods In this study, the interaction between nucleic acid bases and a (4, 4) single-walled carbon nanotube (SCNT) were investigated through calculations within quantum mechanics (QM) method at theoretical level of Hartree-Fock (HF) method using 6-31G basis set. Hence, the physical properties such as electronic energy, total dipole moment, charge distributions and binding energy of nucleic acid bases interaction with SCNT were investigated based on HF method. Results It has been found that the guanine base adsorption is bound stronger to the outer surface of nanotube in comparison to the other bases, consistent with the recent theoretical studies. In the other words, the results explored that guanine interaction with SCNT has optimum level of electronic energy so that their interaction is stable. Also, the calculations illustrated that SCNT interact to nucleic acid bases by noncovalent interaction because of charge distribution an electrostatic area is created in place of interaction. Conclusions Consequently, small diameter SCNT interaction with nucleic acid bases is noncovalent. Also, the results revealed that small diameter SCNT interaction especially SCNT (4, 4) with nucleic acid bases can be useful in practical application area of biomedical fields such detection and drug delivery.

  16. Nucleic acids for recognition and catalysis: landmarks, limitations, and looking to the future.

    Science.gov (United States)

    Perrin, D M

    2000-06-01

    Combinatorial selection of nucleic acids has led to the discovery of novel ligands and catalysts that have implications for both chemistry and medicine. In the context of combinatorial chemistry, degenerate syntheses of nucleic acid libraries readily generate as many as 10(15) different molecules in which a small percentage exhibit interesting binding and/or catalytic properties. The primary advantage of nucleic acids is that library coding is an intrinsic property; sequential composition directly determines the activity. At low temperatures, the sequential composition of single stranded nucleic acids governs folding into irregular tertiary structures resulting in interesting activities. At higher temperatures, the same structures are unfolded and decoded by polymerases to reveal sequential information. The use of PCR (polymerase chain reaction) permits amplification and thus enrichment of the selected activity which is then regenerated chemi-enzymatically. Iterative selection and amplification result in one of the highest throughput screens conceivable whereby each molecule encodes its own activity permitting the ultimate in parallel sampling. Finally, sequence information, and by extension the chemical composition, is obtained by simple sequencing techniques obviating the need for mass spectrometric deconvolution, parallel tagging, and/or large volumes needed for viral and cell culture. This review begins with an introduction of general concepts and considerations. The potential for nucleic acids to generate tight-binding ligands is of interest to structural biologists and medicinal chemists. The therapeutic implications to medicine are also touched upon. Since combinatorially selected nucleic acids and antibodies share many conceptual similarities, their respective advantages and limitations are compared. Theoretical and practical limitations for catalyst discovery are discussed along with the use of other chemical and physical approaches to address some current

  17. 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

  18. Structure and behaviour of proteins, nucleic acids and viruses from vibrational Raman optical activity

    DEFF Research Database (Denmark)

    Barron, L.D.; Blanch, E.W.; McColl, I.H.; Syme, C.D.; Hecht, L.; Nielsen, Kurt

    stacking arrangement and the mutual orientation of the sugar and base rings around the C-N glycosidic link. The ROA spectra of intact viruses provide information on the folds of the coat proteins and the nucleic acid structure. The large number of structure-sensitive bands in protein ROA spectra is...... aqueous solution. Protein ROA spectra provide information on the secondary and tertiary structure of the polypeptide backbone, hydration, side chain conformation and structural elements present in denatured states. Nucleic acid ROA spectra provide information on the sugar ring conformation, the base...

  19. Effect of storage of rice seeds on solute leaching and nucleic acid synthesis

    International Nuclear Information System (INIS)

    Rice (Oryza sativa Linn.) seeds of early varieties 'Ratna' and 'Jaya' and late varieties 'Suakalma' and 'Pankaj' remained viable for 12 to 16 months in normal storage. Conductivity of pooled leachates, leaching of soluble carbohydrate and nitrogen increased with duration of storage. While 32P uptake in isolated embryos and endosperms of fresh, 1-year and 2-year-old seeds showed a declining trend, its incorporation in nucleic acids did not vary significantly. The content of nucleic acid of seeds, however, decreased with an increase in the storage period. (auth.)

  20. Sequence-specific nucleic acid detection from binary pore conductance measurement

    OpenAIRE

    Esfandiari, Leyla; Monbouquette, Harold G.; Jacob J. Schmidt

    2012-01-01

    We describe a platform for sequence-specific nucleic acid (NA) detection utilizing a micropipette tapered to a 2 μm diameter pore and 3 μm diameter polystyrene beads to which uncharged peptide nucleic acid (PNA) probe molecules have been conjugated. As the target NAs hybridize to the complementary PNA-beads, the beads acquire negative charge and become electrophoretically mobile. An applied electric field guides these NA-PNA-beads toward the pipette tip, which they obstruct, leading to an ind...

  1. 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...

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

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Deborggraeve, Stijn; Büscher, Philippe; Nielsen, Peter E

    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...

  3. Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

    Directory of Open Access Journals (Sweden)

    Carl A. Batt

    2009-05-01

    Full Text Available The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform.

  4. Real-Time PCR for detection of herpes simplex virus without nucleic acid extraction

    Directory of Open Access Journals (Sweden)

    Klausner Jeffrey

    2006-06-01

    Full Text Available Abstract Background The speed and sensitivity of real-time polymerase chain reaction (PCR have made it a popular method for the detection of microbiological agents in both research and clinical specimens. For the detection and genotyping of herpes simplex virus (HSV in clinical specimens, real-time PCR has proven to be faster, more sensitive and safer than earlier methods which included isolation of the virus in cell culture followed by immunofluorescence microscopy. While PCR-based assays for HSV detection posses clear advantages over these earlier techniques, certain aspects of the PCR method remain onerous. The process of extraction and purification of nucleic acid from clinical specimens prior to PCR is particularly cumbersome. Nucleic acid extraction is expensive, time-consuming and provides a step whereby specimens can become contaminated prior to their analysis. Herein, we investigate the necessity of nucleic acid extraction from swab-based clinical specimens for HSV detection by real-time PCR. We find that nucleic acid extraction is unnecessary for specific and sensitive detection of HSV in clinical specimens using real-time PCR. Methods Prospective (n = 36 and retrospective (n = 21 clinical specimens from various anatomical sites were analyzed for the presence of herpes simplex virus 1 or 2 by real-time PCR using the RealArt HSV 1/2 LC PCR Kit. Specimens were analyzed by PCR both before and following automated nucleic acid extraction. PCR using extracted and unextracted specimens was also compared to cell culture as a means of detecting HSV. Results Detection of HSV 1/2 DNA in clinical specimens by real-time PCR did not require that the specimen be subjected to nucleic acid extraction/purification prior to analysis. Each specimen that was detectable by real-time PCR when analyzed in the extracted form was also detectable when analyzed in the unextracted form using the methods herein. The limit of detection of HSV-1 and HSV-2 particles

  5. Locked nucleic acid (LNA): High affinity targeting of RNA for diagnostics and therapeutics

    DEFF Research Database (Denmark)

    Kauppinen, S.; Vester, Birte; Wengel, Jesper

    2005-01-01

    Locked nucleic acid (LNA) is a nucleic acid analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation. This conformational restriction results in unprecedented hybridization affinity towards complementary single stranded RNA...... and thus, makes LNA uniquely suited for mimicking RNA structures and sequence specific targeting of RNA in vitro or in vivo. The focus of this paper is on LNAantisense, LNA-modified siRNA (siLNA), and detection and analysis of microRNAs by LNA-modified oligonucleotide probes....

  6. 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. PMID:26503258

  7. 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.

  8. 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; Schmidt, Steffen; Møller, Jesper Bonnet; Holmskov, Uffe; Mollenhauer, Jan

    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 ...

  9. 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 diseases... (.csml) Show Plasmacytoid dendritic cells: sensing nucleic acids in viral infection andautoimmune diseases....iral infection andautoimmune diseases. Authors Gilliet M, Cao W, Liu YJ. Publication Nat Rev Immunol. 2008 A

  10. 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

    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) str...

  11. Unlocked Nucleic Acids with a Pyrene-Modified Uracil: Synthesis, Hybridization Studies, Fluorescent Properties and i-Motif Stability

    Czech Academy of Sciences Publication Activity Database

    Perlíková, Pavla; Karlsen, K. K.; Pedersen, E. B.; Wengel, J.

    2014-01-01

    Roč. 15, č. 1 (2014), s. 146-156. ISSN 1439-4227 Grant ostatní: European Research Council(XE) FP7-268776 Institutional support: RVO:61388963 Keywords : fluorescence * i-motifs * nucleic acid hybridization * oligonucleotides * unlocked nucleic acids Subject RIV: CE - Biochemistry Impact factor: 3.088, year: 2014

  12. Chemical Architecture and Applications of Nucleic Acid Derivatives Containing 1,2,3-Triazole Functionalities Synthesized via Click Chemistry

    Directory of Open Access Journals (Sweden)

    Wei Gong

    2012-10-01

    Full Text Available There is considerable attention directed at chemically modifying nucleic acids with robust functional groups in order to alter their properties. Since the breakthrough of copper-assisted azide-alkyne cycloadditions (CuAAC, there have been several reports describing the synthesis and properties of novel triazole-modified nucleic acid derivatives for potential downstream DNA- and RNA-based applications. This review will focus on highlighting representative novel nucleic acid molecular structures that have been synthesized via the “click” azide-alkyne cycloaddition. Many of these derivatives show compatibility for various applications that involve enzymatic transformation, nucleic acid hybridization, molecular tagging and purification, and gene silencing. The details of these applications are discussed. In conclusion, the future of nucleic acid analogues functionalized with triazoles is promising.

  13. Determination of the Nucleic Acid Adducts Structure at the Nucleoside/Nucleotide Level by NMR Spectroscopy

    Czech Academy of Sciences Publication Activity Database

    Dračínský, Martin; Pohl, Radek

    2015-01-01

    Roč. 28, č. 2 (2015), s. 155-165. ISSN 0893-228X R&D Projects: GA ČR GA13-24880S Institutional support: RVO:61388963 Keywords : NMR spectroscopy * nucleic acids * nucleotides Subject RIV: CC - Organic Chemistry Impact factor: 3.529, year: 2014

  14. 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...

  15. Studies on the effect of organophosphorus insecticide (dichlorovos) on nucleic acids and its toxicity on mice

    International Nuclear Information System (INIS)

    The effect of organophosphorus insecticide dichlorovos on nucleic acids and its toxicity in mice was investigated. The studies carried out included distribution study of the insecticide in different organs of mice, its effect on acetyl cholinesterase both on blood and in brain and alkylation capability on guanine in urine and in intact DNA of mouse liver

  16. 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. PMID:8536076

  17. Nucleic Acids and Enzymes at Electrodes: Electrochemical Nanomedical Biosensors and Biofuel Cell Development

    DEFF Research Database (Denmark)

    Ferapontova, Elena

    for nanomedicine, based on DNA and RNA architectures (1, 4, 5), in which binding of the analyte results in the electrochemically translatable conformational nanoswitching of nucleic acids, with a special emphasis on electronic molecular beacon systems for genetic and small-molecule electroanalysis. Future...

  18. Fluorescence Quenching Investigation for Janus Green B and used as Probe in Determination of Nucleic Acids

    Institute of Scientific and Technical Information of China (English)

    陈莉华; 刘六战; 沈含熙

    2005-01-01

    Fluorescence quenching of janus green B (JGB) in sodium dodecyl sulfate (SDS) micelle by nucleic acids (DNA) was studied using UV-vis absorption, steady state fluorescence emission methods and lifetime measurements. In the SDS micelle, weak fluorescence of JGB was enhanced, and the maximum emission shifted from 425 to 410 nm. In the presence of DNA, the fluorescence of JGB was quenched. Linear relationships between the fluorescence quenching (F0/F) and concentrations of DNA were observed in the range of 2.4×10-8 to 1.08×10-7mol·L-1 for calf thymus nucleic acids (ct DNA) and 1.9×10-8 to 3.8×10-8 mol·L-1 for fish sperm nucleic acids (fs DNA) when 2.5×10-5 mol·L-1 JGB was employed. The limit detection were 1.3×10-8 mol·L-1 for ct DNA and 6.4×10-9 mol·L-1 for fs DNA. At high DNA concentration, there was a systematic deviation from the Stem-Volmer equation due to the static and dynamic quenching occurring simultaneously. The proposed method was applied to the determination of the nucleic acids in chicken blood extraction and the analytical results were in good agreement with the UV-method.

  19. Polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics.

    Science.gov (United States)

    Linnes, J C; Rodriguez, N M; Liu, L; Klapperich, C M

    2016-04-01

    Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications. PMID:26906904

  20. Flexibility-rigidity index for protein-nucleic acid flexibility and fluctuation analysis.

    Science.gov (United States)

    Opron, Kristopher; Xia, Kelin; Burton, Zach; Wei, Guo-Wei

    2016-05-30

    Protein-nucleic acid complexes are important for many cellular processes including the most essential functions such as transcription and translation. For many protein-nucleic acid complexes, flexibility of both macromolecules has been shown to be critical for specificity and/or function. The flexibility-rigidity index (FRI) has been proposed as an accurate and efficient approach for protein flexibility analysis. In this article, we introduce FRI for the flexibility analysis of protein-nucleic acid complexes. We demonstrate that a multiscale strategy, which incorporates multiple kernels to capture various length scales in biomolecular collective motions, is able to significantly improve the state of art in the flexibility analysis of protein-nucleic acid complexes. We take the advantage of the high accuracy and O(N) computational complexity of our multiscale FRI method to investigate the flexibility of ribosomal subunits, which are difficult to analyze by alternative approaches. An anisotropic FRI approach, which involves localized Hessian matrices, is utilized to study the translocation dynamics in an RNA polymerase. © 2016 Wiley Periodicals, Inc. PMID:26927815

  1. Nuclemeter: A Reaction-Diffusion Column for Quantifying Nucleic Acids Undergoing Enzymatic Amplification

    Science.gov (United States)

    Bau, Haim; Liu, Changchun; Killawala, Chitvan; Sadik, Mohamed; Mauk, Michael

    2014-11-01

    Real-time amplification and quantification of specific nucleic acid sequences plays a major role in many medical and biotechnological applications. In the case of infectious diseases, quantification of the pathogen-load in patient specimens is critical to assessing disease progression, effectiveness of drug therapy, and emergence of drug-resistance. Typically, nucleic acid quantification requires sophisticated and expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low resource settings. We describe a simple, low-cost, reactiondiffusion based method for end-point quantification of target nucleic acids undergoing enzymatic amplification. The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer. We model the process with the Fisher Kolmogoroff Petrovskii Piscounoff (FKPP) Equation and compare theoretical predictions with experimental observations. The proposed method is suitable for nucleic acid quantification at the point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free. C.L. was supported by NIH/NIAID K25AI099160; M.S. was supported by the Pennsylvania Ben Franklin Technology Development Authority; C.K. and H.B. were funded, in part, by NIH/NIAID 1R41AI104418-01A1.

  2. Chemical Shifts in Nucleic Acids Studied by Density Functional Theory Calculations and Comparison with Experiment

    Czech Academy of Sciences Publication Activity Database

    Fonville, J. M.; Swart, M.; Vokáčová, Zuzana; Sychrovský, Vladimír; Šponer, Judit E.; Šponer, Jiří; Hilbers, C. W.; Bickelhaupt, F. M.; Wijmenga, S. S.

    2012-01-01

    Roč. 18, č. 39 (2012), s. 12372-12387. ISSN 0947-6539 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040702; CEZ:AV0Z50040507 Keywords : density functional calculations * NMR spectroscopy * nucleic acids * structure elucidation Subject RIV: CF - Physical ; Theoretical Chemistry; BO - Biophysics (BFU-R) Impact factor: 5.831, year: 2012

  3. Nuclemeter: a reaction-diffusion based method for quantifying nucleic acids undergoing enzymatic amplification.

    Science.gov (United States)

    Liu, Changchun; Sadik, Mohamed M; Mauk, Michael G; Edelstein, Paul H; Bushman, Frederic D; Gross, Robert; Bau, Haim H

    2014-01-01

    Real-time amplification and quantification of specific nucleic acid sequences plays a major role in medical and biotechnological applications. In the case of infectious diseases, such as HIV, quantification of the pathogen-load in patient specimens is critical to assess disease progression and effectiveness of drug therapy. Typically, nucleic acid quantification requires expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low-resource settings. This paper describes a simple, low-cost, reaction-diffusion based method for end-point quantification of target nucleic acids undergoing enzymatic amplification. The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer. The method was tested for HIV viral load monitoring and performed on par with conventional benchtop methods. The proposed method is suitable for nucleic acid quantification at point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free. PMID:25477046

  4. 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

    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 and...

  5. Free radical damage of nucleic acids and their components. I. Direct absorption of energy

    International Nuclear Information System (INIS)

    An attempt is made to summarize our present understanding of free radical formation and reactions when energy from ionizing radiations is deposited directly in nucleic acids. The scope of the discussion is limited to recent progress, with reference to older work only when needed for coherence

  6. Recognition of binding sites and targeting of drugs on nucleic acids

    Czech Academy of Sciences Publication Activity Database

    Šíp, Miroslav

    České Budějovice : Kopp Publishing, 2002 - (Berger, J.), s. 84-85 [Conference on Cell Biology /4./. České Budějovice (CZ), 09.09.2002-11.09.2002] Institutional research plan: CEZ:AV0Z5051902 Keywords : nucleic acid * binding sites Subject RIV: BO - Biophysics

  7. Substitution-Inert Trinuclear Platinum Complexes Efficiently Condense/Aggregate Nucleic Acids and Inhibit Enzymatic Activity

    Czech Academy of Sciences Publication Activity Database

    Malina, Jaroslav; Farrell, N. P.; Brabec, Viktor

    2014-01-01

    Roč. 53, č. 47 (2014), s. 12812-12816. ISSN 1433-7851 R&D Projects: GA ČR(CZ) GA13-08273S Institutional support: RVO:68081707 Keywords : DNA condensation * nucleic acids * platinum Subject RIV: BO - Biophysics Impact factor: 11.261, year: 2014

  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...... step is a pre-requisite for performing LNA-modified RNA aptamer selection....

  9. Identification of Low-Molecular-Weight Nucleic Acid-Related Substances Secreted by Streptomyces aureofaciens

    OpenAIRE

    De Carvalho, Alírio; Molinari, Rubens

    1983-01-01

    Streptomyces aureofaciens growth in chemically defined medium is actively associated with the secretion of low-molecular-weight nucleic acid-related substances and is linked to low availability of phosphate. Thirteen pure compounds were isolated, of which seven were identified.

  10. Amplified electrochemical detection of nucleic acid hybridization via selective preconcentration of unmodified gold nanoparticles.

    Science.gov (United States)

    Li, Yuan; Tian, Rui; Zheng, Xingwang; Huang, Rongfu

    2016-08-31

    The common drawback of optical methods for rapid detection of nucleic acid by exploiting the differential affinity of single-/double-stranded nucleic acids for unmodified gold nanoparticles (AuNPs) is its relatively low sensitivity. In this article, on the basis of selective preconcentration of AuNPs unprotected by single-stranded DNA (ssDNA) binding, a novel electrochemical strategy for nucleic acid sequence identification assay has been developed. Through detecting the redox signal mediated by AuNPs on 1, 6-hexanedithiol blocked gold electrode, the proposed method is able to ensure substantial signal amplification and a low background current. This strategy is demonstrated for quantitative analysis of the target microRNA (let-7a) in human breast adenocarcinoma cells, and a detection limit of 16 fM is readily achieved with desirable specificity and sensitivity. These results indicate that the selective preconcentration of AuNPs for electrochemical signal readout can offer a promising platform for the detection of specific nucleic acid sequence. PMID:27506344

  11. 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.

  12. 78 FR 16513 - Application of Advances in Nucleic Acid and Protein Based Detection Methods to Multiplex...

    Science.gov (United States)

    2013-03-15

    ... workshop are to review the status of multiplex platforms and the technological advances in gene based and... academic institutions, blood establishments, industry, and government agencies. Date and Time: The public... HUMAN SERVICES Food and Drug Administration Application of Advances in Nucleic Acid and Protein...

  13. Nucleic acid probes as a diagnostic method for tick-borne hemoparasites of veterinary importance.

    Science.gov (United States)

    Figueroa, J V; Buening, G M

    1995-03-01

    An increased number of articles on the use of nucleic acid-based hybridization techniques for diagnostic purposes have been recently published. This article reviews nucleic acid-based hybridization as an assay to detect hemoparasite infections of economic relevance in veterinary medicine. By using recombinant DNA techniques, selected clones containing inserts of Anaplasma, Babesia, Cowdria or Theileria genomic DNA sequences have been obtained, and they are now available to be utilized as specific, highly sensitive DNA or RNA probes to detect the presence of the hemoparasite DNA in an infected animal. Either in an isotopic or non-isotopic detection system, probes have allowed scientists to test for--originally in samples collected from experimentally infected animals and later in samples collected in the field--the presence of hemoparasites during the prepatent, patent, convalescent, and chronic periods of the infection in the host. Nucleic acid probes have given researchers the opportunity to carry out genomic analysis of parasite DNA to differentiate hemoparasite species and to identify genetically distinct populations among and within isolates, strains and clonal populations. Prevalence of parasite infection in the tick vector can now be accomplished more specifically with the nucleic acid probes. Lately, with the advent of the polymerase chain reaction technique, small numbers of hemoparasites can be positively identified in the vertebrate host and tick vector. These techniques can be used to assess the veterinary epidemiological situation in a particular geographical region for the planning of control measures. PMID:7597795

  14. Comparison of three methods for isolation of nucleic acids from membranate inner ear tissue of rats

    Institute of Scientific and Technical Information of China (English)

    KONG Wei-jia; WANG Ying; WANG Qiong; HAN Yue-chen; HU Yu-juan

    2006-01-01

    Background Mitochondrial DNA mutations have been found in sensorineural deafness. The aim of this study was to compare three methods for extraction of nucleic acid from membranate inner ear tissue of rats.Methods Alkaline denaturation, a conventional phenol-chloroform method and Trizol reagent were respectively used to extract the slight nucleic acid from membranate inner ear tissue of rats. We assessed the amount and quality of nucleic acid using a UV-spectrometer and polymerase chain reaction (PCR).Results The yield and purity (OD260/OD280) of DNA from inner ear tissue using the phenol-chloroform method was the highest of the three methods. Mitochondrial DNA (mtDNA) fragment can be amplified by PCR from nucleic acid prepared by all methods, while no nuclear DNA (nDNA) fragment can be amplified by method of alkaline denaturation. Both nuclear and mitochondrial genescould be amplified by reverse transcriptional PCR from the RNA prepared by Trizol reagent.Conclusion Adequate amount and high-quality of mtDNA, nDNA and RNA were obtained from unilateral membranate inner ear tissue of rats. Method of alkaline denaturation could be chosen when mtDNA without nDNA was needed, while phenol-chloroform method was suitable for extracting total DNA (including nDNA and mtDNA); method with Trizol reagent was suitable for extracting total RNA and total DNA.

  15. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions.

    Science.gov (United States)

    Vickers, Timothy A; Crooke, Stanley T

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  16. 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

  17. Label-free direct surface-enhanced Raman scattering (SERS) of nucleic acids (Conference Presentation)

    Science.gov (United States)

    Guerrini, Luca; Morla-Folch, Judit; Gisbert-Quilis, Patricia; Xie, Hainan; Alvarez-Puebla, Ramon

    2016-03-01

    Recently, plasmonic-based biosensing has experienced an unprecedented level of attention, with a particular focus on the nucleic acid detection, offering efficient solutions to engineer simple, fast, highly sensitive sensing platforms while overcoming important limitations of PCR and microarray techniques. In the broad field of plasmonics, surface-enhanced Raman scattering (SERS) spectroscopy has arisen as a powerful analytical tool for detection and structural characterization of biomolecules. Today applications of SERS to nucleic acid analysis largely rely on indirect strategies, which have been demonstrated very effective for pure sensing purposes but completely dismiss the exquisite structural information provided by the direct acquisition of the biomolecular vibrational fingerprint. Contrarily, direct label-free SERS of nucleic acid shows an outstanding potential in terms of chemical-specific information which, however, remained largely unexpressed mainly because of the inherent poor spectral reproducibility and/or limited sensitivity. To address these limitations, we developed a fast and affordable high-throughput screening direct SERS method for gaining detailed genomic information on nucleic acids (DNA and RNA) and for the characterization and quantitative recognition of DNA interactions with exogenous agents. The simple strategy relies on the electrostatic adhesion of DNA/RNA onto positively-charged silver colloids that promotes the nanoparticle aggregation into stable clusters yielding intense and reproducible SERS spectra at picogram level (i.e. the analysis can be performed without the necessity of amplification steps thus providing realistic direct information of the nucleic acid in its native state). We anticipate this method to gain a vast impact and set of applications in different fields, including medical diagnostics, genomic screening, drug discovery, forensic science and even molecular electronics.

  18. 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.

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

    International Nuclear Information System (INIS)

    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

  20. RNA:DNA Ratio and Other Nucleic Acid Derived Indices in Marine Ecology

    Directory of Open Access Journals (Sweden)

    Luis Chícharo

    2008-08-01

    Full Text Available Some of most used indicators in marine ecology are nucleic acid-derived indices. They can be divided by target levels in three groups: 1 at the organism level as ecophysiologic indicators, indicators such as RNA:DNA ratios, DNA:dry weight and RNA:protein, 2 at the population level, indicators such as growth rate, starvation incidence or fisheries impact indicators, and 3 at the community level, indicators such as trophic interactions, exergy indices and prey identification. The nucleic acids derived indices, especially RNA:DNA ratio, have been applied with success as indicators of nutritional condition, well been and growth in marine organisms. They are also useful as indicators of natural or anthropogenic impacts in marine population and communities, such as upwelling or dredge fisheries, respectively. They can help in understanding important issues of marine ecology such as trophic interactions in marine environment, fish and invertebrate recruitment failure and biodiversity changes, without laborious work of counting, measuring and identification of small marine organisms. Besides the objective of integrate nucleic acid derived indices across levels of organization, the paper will also include a general characterization of most used nucleic acid derived indices in marine ecology and also advantages and limitations of them. We can conclude that using indicators, such RNA:DNA ratios and other nucleic acids derived indices concomitantly with organism and ecosystems measures of responses to climate change (distribution, abundance, activity, metabolic rate, survival will allow for the development of more rigorous and realistic predictions of the effects of anthropogenic climate change on marine systems.

  1. Nanopore Analysis of Nucleic Acids: Single-Molecule Studies of Molecular Dynamics, Structure, and Base Sequence

    Science.gov (United States)

    Olasagasti, Felix; Deamer, David W.

    Nucleic acids are linear polynucleotides in which each base is covalently linked to a pentose sugar and a phosphate group carrying a negative charge. If a pore having roughly the crosssectional diameter of a single-stranded nucleic acid is embedded in a thin membrane and a voltage of 100 mV or more is applied, individual nucleic acids in solution can be captured by the electrical field in the pore and translocated through by single-molecule electrophoresis. The dimensions of the pore cannot accommodate anything larger than a single strand, so each base in the molecule passes through the pore in strict linear sequence. The nucleic acid strand occupies a large fraction of the pore's volume during translocation and therefore produces a transient blockade of the ionic current created by the applied voltage. If it could be demonstrated that each nucleotide in the polymer produced a characteristic modulation of the ionic current during its passage through the nanopore, the sequence of current modulations would reflect the sequence of bases in the polymer. According to this basic concept, nanopores are analogous to a Coulter counter that detects nanoscopic molecules rather than microscopic [1,2]. However, the advantage of nanopores is that individual macromolecules can be characterized because different chemical and physical properties affect their passage through the pore. Because macromolecules can be captured in the pore as well as translocated, the nanopore can be used to detect individual functional complexes that form between a nucleic acid and an enzyme. No other technique has this capability.

  2. Study of nucleic acids variations in children in nearest areas to the Chernobyl accident

    International Nuclear Information System (INIS)

    The technique used to determine the nucleic acids concentration of leucocytes in peripheral blood is simple, quick and reproducible. Mathematical patterns for small doses have been achieved by means of this technique, which is also useful as biochemical indicator for evaluating exposure to ionizing radiations. The following information is the result of a research in 445 children from 43 locations that suffered, in different degrees, the effect of this accident. Children were grouped taking into account different degrees of superficial pollution for Cs-137 of original places. Groups were built as follows: G-1, 165 children from 20 superficially polluted places between 0-37 KBq/m2; G-2, 135 children from 15 locations with a level of superficial pollution higher than 37 KBq/m2 and lower than 185 KBq/m2; G-3, 85 children from 7 locations with a pollution degree higher than 296 KBq/m2 and G-4, 60 children from a place with a non-determined superficial pollution. Values within the interval 1,29 - 4,89 mg/100 ml of blood samples taken from healthy children in group G-1 were considered as normal. The increase in dose led neither to a decrease in nucleic acids medium value, nor to a meaningful growth in the number of cases with nucleic acids figures under rank considered normal. On the other hand, thyroid hyperplasia did not lead either to an increase in medium values of nucleic acids or to an increase in the number of cases with nucleic acids figures over intervals considered as normal. (authors). 10 refs., 2 tabs

  3. A model for protocellular coordination of nucleic acid and protein syntheses

    Science.gov (United States)

    Fox, S. W.

    1981-01-01

    The proteinoid model for the coordination of protein synthesis with nucleic acid coding within the evolving protocell is discussed. Evidence for the self-ordering of amino acid chains, which would enhance the catalytic activity of a lysine-rich proteinoid, is presented, along with that for the preferential formation of microparticles, particularly proteinoid microparticles, in various solutions. Demonstrations of the catalytic activity of lysine-rich proteinoids in the synthesis of peptide and internucleotide bonds are pointed out. The view of evolution as a two stage sequence in which the geological synthesis of peptides evolved to the protocellular synthesis of peptides and oligonucleotides is discussed, and contrasted with the alternative view, in accord with the central dogma, that nucleic acids arose first then governed the production of proteins and protocells.

  4. Tunnel effect in excited and ionized states of nucleic acid bases and some aspects of radiation-induced point gene mutations

    International Nuclear Information System (INIS)

    Radiation induced perturbations of the genetic code are discussed from the standpoint of the frequency and specificity of mutations. According to Lowdin's theory of tautomeric rearrangement of nucleic acid base pairs through the tunnel effect, it is probable, that the proton potential in hydrogen bridges can be also effected by the incorporation of some radiolytic products of purines and pyrimidines into DNA as mistake bases. In this way it is possible, to eliminate any exo-or endogeneous energetic irradiation of the biological material and so to eliminate various undesirable damages of DNA. Thus higher specificity in the controlling of the genetic code changes would result. (F.G.)

  5. Design and Synthesis of Novel Peptide Nucleic Acid Monomers

    Institute of Scientific and Technical Information of China (English)

    白金泉; 李英; 刘克良

    2001-01-01

    All of the four nucleobases in DNA have replaced the 4-hydroxy group of N-[2-(tert-butoxycarbonylaminomethyl)-trams-4-hydroxy]tetrahydropyrrole acetic acid methyl ester with cis-stereochemistry. An efficient route for the synthesis of N-[2-(tert-butoxycarbonylaminomethyl)-trans-4-hydroxy]-tetrahydropyrrole acetic acid methyl ester has been developed.Starting with this intermediate, the protected monmers were synthesized by the Mitsunobu reaction or via its tosylate.

  6. Nucleic acid fragmentation on the millisecond timescale using a conventional X-ray rotating anode source: application to protein–DNA footprinting

    OpenAIRE

    Henn, Arnon; Halfon, Jacob; Kela, Itai; Orion, Itzhak; Sagi, Irit

    2001-01-01

    Nucleic acid fragmentation (footprinting) by ·OH radicals is used often as a tool to probe nucleic acid structure and nucleic acid–protein interactions. This method has proven valuable because it provides structural information with single base pair resolution. Recent developments in the field introduced the ‘synchrotron X-ray footprinting’ method, which uses a high-flux X-ray source to produce single base pair fragmentation of nucleic acid in tens of milliseconds. We developed a complementar...

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

    International Nuclear Information System (INIS)

    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. The online version of this article (doi:10.1186/s13628-015-0019-5) contains supplementary material, which

  8. 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

  9. Nucleic Acid Analogue Induced Transcription of Double Stranded DNA

    DEFF Research Database (Denmark)

    1998-01-01

    RNA is transcribed from a double stranded DNA template by forming a complex by hybridizing to the template at a desired transcription initiation site one or more oligonucleic acid analogues of the PNA type capable of forming a transcription initiation site with the DNA and exposing the complex to...... displacement of one strand of the DNA locally by the PNA hybridization....

  10. 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

    Nucleic acid aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with very high affinity and specificity, and are generally selected by a process referred to as systematic evolution of ligands by exponential enrichment. Conventional antibody-based therape...... 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.......-based therapeutic and diagnostic approach currently employed against biotoxins pose major limitations such as the requirement of a live animal for the in vivo enrichment of the antibody species, decreased stability, high production cost, and side effects. Aptamer technology is a viable alternative that can be used...

  11. 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.

  12. Safety profile of the intravenous administration of brain-targeted stable nucleic acid lipid particles.

    Science.gov (United States)

    Conceição, Mariana; Mendonça, Liliana; Nóbrega, Clévio; Gomes, Célia; Costa, Pedro; Hirai, Hirokazu; Moreira, João Nuno; Lima, Maria C; Manjunath, N; de Almeida, Luís Pereira

    2016-03-01

    In a clinical setting, where multiple administrations of the therapeutic agent are usually required to improve the therapeutic outcome, it is crucial to assess the immunogenicity of the administered nanoparticles. In this data work, we investigated the safety profile of the repeated intravenous administration of brain-targeted stable nucleic acid lipid particles (RVG-9r-targeted SNALPs). To evaluate local activation of the immune system, we performed analysis of mouse tissue homogenates and sections from cerebellum. To investigate peripheral activation of the immune system, we used serum of mice that were intravenously injected with RVG-9r-targeted SNALPs. These data are related and were discussed in the accompanying research article entitled "Intravenous administration of brain-targeted stable nucleic acid lipid particles alleviates Machado-Joseph disease neurological phenotype" (Conceição et al., in press) [1]. PMID:26958628

  13. Isothermal amplification detection of nucleic acids by a double-nicked beacon.

    Science.gov (United States)

    Shi, Chao; Zhou, Meiling; Pan, Mei; Zhong, Guilin; Ma, Cuiping

    2016-03-01

    Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use. PMID:26706801

  14. Saccharides as Prospective Immobilizers of Nucleic Acids for Room-Temperature Structural EPR Studies.

    Science.gov (United States)

    Kuzhelev, Andrey A; Shevelev, Georgiy Yu; Krumkacheva, Olesya A; Tormyshev, Victor M; Pyshnyi, Dmitrii V; Fedin, Matvey V; Bagryanskaya, Elena G

    2016-07-01

    Pulsed dipolar electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for structural studies of biomolecules and their complexes. This method, whose applicability has been recently extended to room temperatures, requires immobilization of the studied biosystem to prevent averaging of dipolar couplings; at the same time, the modification of native conformations by immobilization must be avoided. In this work, we provide first demonstration of room-temperature EPR distance measurements in nucleic acids using saccharides trehalose, sucrose, and glucose as immobilizing media. We propose an approach that keeps structural conformation and unity of immobilized double-stranded DNA. Remarkably, room-temperature electron spin dephasing time of triarylmethyl-labeled DNA in trehalose is noticeably longer compared to previously used immobilizers, thus providing a broader range of available distances. Therefore, saccharides, and especially trehalose, can be efficiently used as immobilizers of nucleic acids, mimicking native conditions and allowing wide range of structural EPR studies at room temperatures. PMID:27320083

  15. 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.

  16. 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. PMID:27030641

  17. Summarization on the synthesis and radionuclide-labeling of peptide nucleic acid for an oligonucleotide analogue

    International Nuclear Information System (INIS)

    Peptide nucleic acid (PNA), which is one kind of antisense nucleic acid compounds and an oligonucleotide analogue that binds strongly to DNA and RNA in a sequence specific manner, has its unique advantages in the field of molecular diagnostics and treatment of diseases. Now, people gradually attach more importance to PNA. To optimize the application of PNA in genetic re- search and therapy, a great number of backbone modifications on the newly- type structures of PNA were synthesized to improve its physicochemical proper- ties, such as hybridization speciality, solubility in biofluid, or cell permeability. The modified PNA labeled with radionuclides, which can obtain the aim at specific target and minimal non-target trauma, has important role in research and application of tumorous genitherapy. Here a review on the basic synthesis idea and several primary synthetic methods of PNA analogs was given, and also correlative studies and expectation on the compounds belonging to PNA series labeled with radionuclides were included. (authors)

  18. 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....... The largest destabilization is observed for position C14, while UNA placed in position A12 causes significant increase of i-DNA thermodynamic stability. CD curves of UNA-modified variants imply no structural changes relative to the native i-motif.......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...

  19. 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.; Belak, S.; Wakeley, P. R.; Reid, S. M.; Ebert, K.; King, D. 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...

  20. Amino-modified tetraphenylethene derivatives as nucleic acid stain: relationship between the structure and sensitivity.

    Science.gov (United States)

    Xu, Li; Zhu, Zece; Wei, Danqing; Zhou, Xiang; Qin, Jingui; Yang, Chuluo

    2014-10-22

    A series of new amino-functionalized tetraphenylethene (TPE) derivatives were designed and synthesized to study the effect of molecular structures on the detection of nucleic acid. Contrastive studies revealed that the number of binding groups, the length of hydrophobic linking arm and the configuration of TPE molecule all play important roles on the sensitivity of the probes in nucleic acid detection. Z-TPE3 with two binding amino groups, long linking arms, and cis configuration was found to be the most sensitive dye in both solution and gel matrix. Z-TPE3 is able to stain dsDNA with the lowest amount of 1 ng and exclusively stain 40 ng of short oligonucleotide with only 10 nt. This work is of important significance for the further design of TPE probes as biosensors with higher sensitivity. PMID:25279446

  1. EFFECT OF SURFACTANT SDS ON DETERMINATION OF NUCLEIC ACID WITH TERBIUM (III)FLUO

    Institute of Scientific and Technical Information of China (English)

    WuHui; WanYu

    2002-01-01

    The effect of an anionic surfactant(sodium dodecylsulfate.SDS)on the fluorescence properties of nucleic acid with terbium(III)is studied.Results show that ri-bonucleir acid (RNA)presents fluorescence reaction with Tb(III)directly.but deoxyribonucleic acid(DNA)pre-sents similar fluorescence reaction only after its denatura-tion.In the presence of SDS ,the fluorescence intensity is 4.0 times and 3.5 times greater than that of DNA and RNA without SDS.

  2. Mild Detritylation of Nucleic Acid Hydroxyl Groups by Warming-up

    OpenAIRE

    Salon, Jozef; Zhang, Bo; Huang, Zhen

    2011-01-01

    It is challenging to effectively deprotect hydroxyl groups of acid-or-base sensitive bio-macromolecules without causing even minor defects and compromising high quality of final products. We report here a mild detritylation strategy in mildly acidic buffers to remove the DMTr protection from the 5’-hydroxyl groups of synthetic nucleic acids. The DMTr-groups can be easily and effectively removed at pH 4.5 or 5.0 with slight warming-up (40 °C), offering virtually quantitative deprotection. This...

  3. Non-Enzymatic Copying of Nucleic Acid Templates

    OpenAIRE

    Blain, Jonathan Craig

    2013-01-01

    All known living cells contain a complex set of molecular machinery to support their growth and replication. However, the earliest cells must have been much simpler, consisting of a compartment and a genetic material to allow for Darwinian evolution. To study these intermediates, plausible model `protocells' must be synthesized in the laboratory since no fossils remain. Recent work has shown that fatty acids can self-assemble into vesicles that are able to grow and divide through simple mecha...

  4. Devices and approaches for generating specific high-affinity nucleic acid aptamers

    Science.gov (United States)

    Szeto, Kylan; Craighead, Harold G.

    2014-09-01

    High-affinity and highly specific antibody proteins have played a critical role in biological imaging, medical diagnostics, and therapeutics. Recently, a new class of molecules called aptamers has emerged as an alternative to antibodies. Aptamers are short nucleic acid molecules that can be generated and synthesized in vitro to bind to virtually any target in a wide range of environments. They are, in principal, less expensive and more reproducible than antibodies, and their versatility creates possibilities for new technologies. Aptamers are generated using libraries of nucleic acid molecules with random sequences that are subjected to affinity selections for binding to specific target molecules. This is commonly done through a process called Systematic Evolution of Ligands by EXponential enrichment, in which target-bound nucleic acids are isolated from the pool, amplified to high copy numbers, and then reselected against the desired target. This iterative process is continued until the highest affinity nucleic acid sequences dominate the enriched pool. Traditional selections require a dozen or more laborious cycles to isolate strongly binding aptamers, which can take months to complete and consume large quantities of reagents. However, new devices and insights from engineering and the physical sciences have contributed to a reduction in the time and effort needed to generate aptamers. As the demand for these new molecules increases, more efficient and sensitive selection technologies will be needed. These new technologies will need to use smaller samples, exploit a wider range of chemistries and techniques for manipulating binding, and integrate and automate the selection steps. Here, we review new methods and technologies that are being developed towards this goal, and we discuss their roles in accelerating the availability of novel aptamers.

  5. 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.

  6. Nucleic Acid Aptamers as Potential Therapeutic and Diagnostic Agents for Lymphoma

    OpenAIRE

    Shum, Ka-To; Zhou, Jiehua; John J. Rossi

    2013-01-01

    Lymphomas are cancers that arise from white blood cells and usually present as solid tumors. Treatment of lymphoma often involves chemotherapy, and can also include radiotherapy and/or bone marrow transplantation. There is an un-questioned need for more effective therapies and diagnostic tool for lymphoma. Aptamers are single stranded DNA or RNA oligonucleotides whose three-dimensional structures are dictated by their sequences. The immense diversity in function and structure of nucleic acids...

  7. High affinity nucleic acid aptamers for streptavidin incorporated into bi-specific capture ligands

    OpenAIRE

    Tahiri-Alaoui, Abdessamad; Frigotto, Laura; Manville, Nick; Ibrahim, Jamal; Romby, Pascale; James, William

    2002-01-01

    We have isolated 2′-Fluoro-substituted RNA aptamers that bind to streptavidin (SA) with an affinity around 7 ± 1.8 nM, comparable with that of recently described peptide aptamers. Binding to SA was not prevented by prior saturation with biotin, enabling nucleic acid aptamers to form useful ternary complexes. Mutagenesis, secondary structure analysis, ribonuclease footprinting and deletion analysis provided evidence for the essential structural features of SA-binding aptamers. In order to prov...

  8. Design and synthesis of polyacrylamide-based oligonucleotide supports for use in nucleic acid diagnostics.

    OpenAIRE

    Fahy, E.; Davis, G R; DiMichele, L J; Ghosh, S. S.

    1993-01-01

    Polyacrylamide supports, in a range of pore sizes, were investigated as nucleic acid affinity matrices for the detection of target DNA or RNA sequences using a sandwich hybridization format. Bromoacetyl and thiol oligonucleotide derivatives were covalently linked to sulfhydryl- and bromoacetyl-polyacrylamide supports with greater than 95% end-attachment efficiencies. These polyacrylamide-oligonucleotide supports were further derivatized with anionic residues to provide multi-functional suppor...

  9. Differential adsorption of nucleic acid bases: Relevance to the origin of life

    OpenAIRE

    Sowerby, Stephen J.; Cohn, Corey A; Heckl, Wolfgang M.; Holm, Nils G

    2001-01-01

    The adsorption of organic molecules onto the surfaces of inorganic solids has long been considered a process relevant to the origin of life. We have determined the equilibrium adsorption isotherms for the nucleic acid purine and pyrimidine bases dissolved in water on the surface of crystalline graphite. The markedly different adsorption behavior of the bases describes an elutropic series: guanine > adenine > hypoxanthine > thymine > cytosine > uracil. We propose th...

  10. Purification of CREB to Apparent Homogeneity; Removal of Truncation Products and Contaminating Nucleic Acid

    OpenAIRE

    Lopez, Dinaida I.; Mick, Jeanne E.; Nyborg, Jennifer K.

    2007-01-01

    The cAMP response element binding protein (CREB) is a mammalian transcription factor which regulates the expression of many cellular genes. CREB is commonly expressed in E. coli and purified by heat-extraction followed by affinity chromatography. We have discovered that although this purification yields a reasonably pure product which is active in DNA-binding and functional assays, it contains a large amount of nucleic acid as well as CREB truncation products and other polypeptides. Consequen...

  11. Recognition of Double Stranded RNA by Guanidine-Modified Peptide Nucleic Acids (GPNA)

    OpenAIRE

    Gupta, Pankaj; Muse, Oluwatoyosi; Rozners, Eriks

    2011-01-01

    Double helical RNA has become an attractive target for molecular recognition because many non-coding RNAs play important roles in control of gene expression. Recently, we discovered that short peptide nucleic acids (PNA) bind strongly and sequence selectively to a homopurine tract of double helical RNA via triple helix formation. Herein we tested if the molecular recognition of RNA can be enhanced by α-guanidine modification of PNA. Our study was motivated by the discovery of Ly and co-worker...

  12. Well-defined Cationic Shell Crosslinked Nanoparticles for Efficient Delivery of DNA or Peptide Nucleic Acids

    OpenAIRE

    Zhang, Ke; Fang, Huafeng; Gang SHEN; 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 elect...

  13. RNA:DNA Ratio and Other Nucleic Acid Derived Indices in Marine Ecology

    OpenAIRE

    Luis Chícharo; Maria Alexandra Chícharo

    2008-01-01

    Some of most used indicators in marine ecology are nucleic acid-derived indices. They can be divided by target levels in three groups: 1) at the organism level as ecophysiologic indicators, indicators such as RNA:DNA ratios, DNA:dry weight and RNA:protein, 2) at the population level, indicators such as growth rate, starvation incidence or fisheries impact indicators, and 3) at the community level, indicators such as trophic interactions, exergy indices and prey identification. The nu...

  14. 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.

  15. Plant responses against invasive nucleic acids: RNA silencing and its suppression by plant viral pathogens

    OpenAIRE

    Alvarado, Veria; Herman B Scholthof

    2009-01-01

    RNA silencing is a common strategy shared by eukaryotic organisms to regulate gene expression, and also operates as a defense mechanism against invasive nucleic acids such as viral transcripts. The silencing pathway is quite sophisticated in higher eukaryotes but the distinct steps and nature of effector complexes vary between and even within species. To counteract this defense mechanism viruses have evolved the ability to encode proteins that suppress silencing to protect their genomes from ...

  16. TMPyP4, a Stabilizer of Nucleic Acid Secondary Structure, Is a Novel Acetylcholinesterase Inhibitor

    OpenAIRE

    Fujiwara, Nana; Mazzola, Michael; Cai, Elizabeth; Wang, Meng; Cave, John W.

    2015-01-01

    The porphyrin compound, TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine), is widely used as a photosensitizer and a modulator of nucleic acid secondary structure stability. Our group recently showed in cultured cells and forebrain slice cultures that this compound can also down regulate expression of Tyrosine hydroxylase (Th), which encodes the rate-limiting enzyme in catecholamine biosynthesis, by stabilizing DNA secondary structures in the Th proximal promoter. The current study so...

  17. Effects of Salinity: Calcium Interaction on Growth and Nucleic Acid Metabolism in Five Species of Chenopodiaceae

    OpenAIRE

    ABO-KASSEM, Essam El-Deen Mohaned

    2007-01-01

    Seed germination, seedling growth, and some enzyme activity of nucleic acid metabolism were studied in 5 members of Chenopodiaceae [Beta vulgaris L., Chenopodium quinoa Willd., Spinacea oleracea L., Allenrolfia occidentalis (S.Watson) Kuntze, Atriplex hortensis L.] under NaCl salinity alone or combined with 0.5 mM CaSO4. High salinity delayed radical emergence and decreased germination percentage in all plants. Combined CaSO4 reduced inhibition of seed germination in B. vulgaris, S. oleracea,...

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

    OpenAIRE

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

    2015-01-01

    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 wit...

  19. DNA-Templated Polymerization of Side-Chain-Functionalized Peptide Nucleic Acid Aldehydes

    OpenAIRE

    Kleiner, Ralph E.; Brudno, Yevgeny; Birnbaum, Michael E.; Liu, David R.

    2008-01-01

    The DNA-templated polymerization of synthetic building blocks provides a potential route to the laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated polymerization of side-chain-functionalized PNA tetramer and pentamer aldehydes. We obs...

  20. A possible mode of the specifi-crecognition of nucleic acids by proteins

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Seven sets of protein target sites, which occur in several gene promoters, have been analyzed. The results suggest that there is a possible mode of specific recognition of double-helical nucleic acids by proteins. This recognition mode is related to a special topological property of double-helical DNA, which is termed base spatial pattern (BSP) of DNA segment. BSP is the spatial topological property determined only by the spatial arrangement of the bases on double-helical DNA segment.

  1. The adaption of an encoded microparticle array for multiplexing nucleic acid hybridisation assays

    OpenAIRE

    Broder, Graham Richard

    2011-01-01

    Our ever increasing knowledge of genetics is radically changing disciplines in science and medicine. Significantly, the study of gene expression and protein synthesis within both healthy and abnormal cells has advanced understanding of the mechanism of disease at the molecular level. The future treatment of certain diseases may benefit from new classes of nucleic acid based drugs which are currently undergoing development and trialling. Concurrently, assays are being formulated...

  2. 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.

  3. End-labeling of peptide nucleic acid with osmium complex. Voltammetry at carbon and mercury electrodes

    Czech Academy of Sciences Publication Activity Database

    Paleček, Emil; Trefulka, Mojmír; Fojta, Miroslav

    2009-01-01

    Roč. 11, č. 2 (2009), s. 359-362. ISSN 1388-2481 R&D Projects: GA AV ČR(CZ) KAN400310651; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : peptide nucleic acid end-labeling * osmium tetroxide complexes * electroactive labels Subject RIV: BO - Biophysics Impact factor: 4.243, year: 2009

  4. Polyanionic Carboxyethyl Peptide Nucleic Acids (ce-PNAs: Synthesis and DNA Binding.

    Directory of Open Access Journals (Sweden)

    Yuliya Kirillova

    Full Text Available New polyanionic modifications of polyamide nucleic acid mimics were obtained. Thymine decamers were synthesized from respective chiral α- and γ-monomers, and their enantiomeric purity was assessed. Here, we present the decamer synthesis, purification and characterization by MALDI-TOF mass spectrometry and an investigation of the hybridization properties of the decamers. We show that the modified γ-S-carboxyethyl-T10 PNA forms a stable triplex with polyadenine DNA.

  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. Evaluation of an Automated Nucleic Acid Extractor for Hepatitis C Virus Load Quantification▿

    OpenAIRE

    Martró, Elisa; García-Sierra, Nerea; González, Victoria; Saludes, Verónica; Matas, Lurdes; Ausina, Vicenç

    2009-01-01

    The increasing use of molecular methods strongly motivates clinical laboratories to introduce automated nucleic acid extractors. We compared the easyMAG (bioMérieux) with a manual extraction method for hepatitis C virus (HCV) load quantification (RealTime HCV; Abbott). Both methods were comparable, and, therefore, the easyMAG is suitable to be implemented in our laboratory for the management of HCV-infected patients.

  7. Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends

    OpenAIRE

    Zhang, Chunsun; Xing, Da

    2007-01-01

    The possibility of performing fast and small-volume nucleic acid amplification and analysis on a single chip has attracted great interest. Devices based on this idea, referred to as micro total analysis, microfluidic analysis, or simply ‘Lab on a chip’ systems, have witnessed steady advances over the last several years. Here, we summarize recent research on chip substrates, surface treatments, PCR reaction volume and speed, architecture, approaches to eliminating cross-contamination and contr...

  8. Effects of Fixation and Storage of Human Tissue Samples on Nucleic Acid Preservation

    OpenAIRE

    Nam, Soo Kyung; Im, Joon; Kwak, Yoonjin; Han, Nayoung; Nam, Kyung Han; Seo, An Na; Lee, Hye Seung

    2014-01-01

    Background Because of recent advances in the molecular diagnosis of cancer patients, tissue quality has become more important in daily practice. Methods To evaluate the effects of fixative, duration of fixation, decalcification, and storage periods on nucleic acid integrity, DNA and RNA were extracted from gastrointestinal cancer tissue. The yield and purity were analyzed, and polymerase chain reaction (PCR) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 60 bp), β-actin (148 bp), and hu...

  9. Nucleic acid detection based on the use of microbeads: a review

    International Nuclear Information System (INIS)

    Microbead-based technologies represent elegant and versatile approaches for highly parallelized quantitative multiparameter assays. They also form the basis of various techniques for detection and quantification of nucleic acids and proteins. Nucleic acid-based methods include hybridization assays, solid-phase PCR, sequencing, and trapping assays. Microbead assays have been improved in the past decades and are now important tools in routine and point-of-care diagnostics as well as in life science. Its advances include low costs, low workload, high speed and high-throughput automation. The potential of microbead-based assays therefore is apparent, and commercial applications can be found in the detection and discrimination of single nucleotide polymorphism, of pathogens, and in trapping assays. This review provides an overview on microbead-based platforms for biosensing with a main focus on nucleic acid detection (including amplification strategies and on selected probe systems using fluorescent labeling). Specific sections cover chemical properties of microbeads, the coupling of targets onto solid surfaces, microbead probe systems (mainly oligonucleotide probes), microbead detection schemes (with subsections on suspension arrays, microfluidic devices, and immobilized microbeads), quantification of nucleic acids, PCR in solution and the detection of amplicons, and methods for solid-phase amplification. We discuss selected trends such as microbead-coupled amplification, heterogeneous and homogenous DNA hybridization assays, real-time assays, melting curve analysis, and digital microbead assays. We finally discuss the relevance and trends of the methods in terms of high-level multiplexed analysis and their potential in diagnosis and personalized medicine. (author)

  10. Electrochemical analysis of nucleic acids and new trends in protein electrochemistry

    Czech Academy of Sciences Publication Activity Database

    Paleček, Emil; Ostatná, Veronika; Trefulka, Mojmír; Černocká, Hana; Bartošík, Martin

    Vienna, 2009. s. 1. ISSN 1091-8213. [216th ECS Meeting with EuroCVD 17 and SOFC XI - 11th International Symposium on Solid Oxide Fuel Cells. 04.10.2009-09.10.2009, Vienna] Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : nucleic acids electrochemistry * protein electrochemistry * chronopotentiometric stripping analysis Subject RIV: BO - Biophysics

  11. 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 ostatní: GA MŠk(CZ) LC512 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

  12. Experimental Warming Decreases the Average Size and Nucleic Acid Content of Marine Bacterial Communities

    Science.gov (United States)

    Huete-Stauffer, Tamara M.; Arandia-Gorostidi, Nestor; Alonso-Sáez, Laura; Morán, Xosé Anxelu G.

    2016-01-01

    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. PMID:27242747

  13. Monoclonal antibodies targeted to alpha-oligonucleotides. Characterisation and application in nucleic acid detection.

    OpenAIRE

    Cros, P.; Kurfürst, R; Allibert, P; Battail, N; Piga, N; Roig, V; Thuong, N T; Mandrand, B; Hélène, C

    1994-01-01

    The aim of the present study was to test the antigenicity of alpha-deoxyribonucleotides in order to develop a new tool for the detection of nucleic acid sequences for use in diagnostic applications. We describe four monoclonal antibodies (Mabs) which recognize alpha-deoxyribonucleotides. Two were raised against a poly(alpha-dT) sequence and specifically recognized the alpha-dT nucleotide. Two were raised against a sequence containing all four common nucleotides as alpha-nucleotides and, surpr...

  14. Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

    OpenAIRE

    Hertoghs, Kirsten M. L.; Ellis, Jonathan H.; Catchpole, Ian R.

    2003-01-01

    The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first ...

  15. 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

    Owing to their constant low temperatures, glacial ice and permafrost might contain the oldest nucleic acids and microbial cells on Earth, which could prove key to reconstructing past ecosystems and for the planning of missions to other planets. However, recent claims concerning viable cells 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...

  16. 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.

  17. Fluorescence Quenching and Binding Interaction of l0-Methylacridinium Iodide to Nucleic Acids

    Institute of Scientific and Technical Information of China (English)

    孙险峰; 江致勤; 丁兵林

    2003-01-01

    Interaction of 10-methylacridinium iodide (MAI) as fluorescence probe with nucleobases, nucleosides and nucleic acids has been studied by UV-visible absorption and fluorescence spectroscopy. It was found that fluorescence of MAI is strongly quenched by the nucleobases, nucleosides and nucleic acids, respectively. The quenching follows the Stern-Volmer linear equation. The fluorescence quenching rate constant (kq) was measured to be 109-1010 (L/mol)/s within the range of diffusion-controlled rate limit, indicating that the interaction between MAI and nucleic acid and their precursors is characteristic of electron transfer mechanism. In addition, the binding interaction model of MAI to calf thymus DNA (ct-DNA) was further investigated. Apparent hypochromism in the absorption spectra of MAI was observed when MAI binds to ct-DNA.Three spectroscopic methods, which include (1) UV spectroscopy, (2) fluorescence quenching of MAI, (3) competitive dual-probe method of MAI and ethidium bromide (EB), were utilized to determine the affinity binding constants (K)of MAI and ct-DNA. The binding constants K obtained from the above methods gave consistent data in the same range (1.0-5.5) ×104 L/mol, which lend credibility to these measurements. The binding site number was determined to be 1.9. The influence of thermal denaturation and phosphate concentration on the binding was examined. The binding model of MAI to ct-DNA including intercalation and outside binding was investigated.

  18. Production of extracellular nucleic acids by genetically altered bacteria in aquatic-environment microcosms

    International Nuclear Information System (INIS)

    The factors which affect the production of extracellular DNA by genetically altered strains of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas cepacia, and Bradyrhizobium japonicum in aquatic environments were investigated. Cellular nucleic acids were labeled in vivo by incubation with [3H]thymidine or [3H]adenine, and production of extracellular DNA in marine waters, artificial seawater, or minimal salts media was determined by detecting radiolabeled macromolecules in incubation filtrates. The presence or absence of the ambient microbial community had little effect on the production of extracellular DNA. Three of four organisms produced the greatest amounts of extracellular nucleic acids when incubated in low-salinity media (2% artificial seawater) rather than high-salinity media (10 to 50% artificial seawater). The greatest production of extracellular nucleic acids by P. cepacia occurred at pH 7 and 37 degree C, suggesting that extracellular-DNA production may be a normal physiologic function of the cell. Incubation of labeled P. cepacia cells in water from Bimini Harbor, Bahamas, resulted in labeling of macromolecules of the ambient microbial population. Collectively these results indicate that (i) extracellular-DNA production by genetically altered bacteria released into aquatic environments is more strongly influenced by physicochemical factors than biotic factors, (ii) extracellular-DNA production rates are usually greater for organisms released in freshwater than marine environments, and (iii) ambient microbial populations can readily utilize materials released by these organisms

  19. Use of radiolabeled nucleic acid probes for the detection and identification of infectious organisms

    International Nuclear Information System (INIS)

    Nucleic acid probes can be used to detect and identify a wide variety of infectious organisms from bacteria to helminthic worms. Commercially-produced kits are now available for the direct detection of Legionella species in clinical samples, for identification of Mycobacterium species growing on solid media or in broth, and for detection of a variety of other bacteria and viruses directly in clinical samples or tissues. 32P and 125I, which are used to label probes to high specific activity, continue to be used in commercially-prepared kits. 32P-labeling remains as one of the most sensitive means of labeling nucleic acids probes. The advantages of using probes to detect and identify infectious agents include the rapid generation of results, the ability to broaden the spectrum of organisms identified by a clinical laboratory, and the high specificity of probe-based tests. The disadvantages of using probes include the high cost of the assays, the additional equipment required, and the inability to perform ancillary tests, such as antimicrobial susceptibility testing, which require having a pure culture of the organism available. None-the-less, nucleic acid probes already are having a very positive impact on the diagnosis of infectious diseases both in the industrialized and developing nations. (author). 11 refs

  20. 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.

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

    Science.gov (United States)

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

    2015-03-01

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

  2. Microfluidic Preparation of Polymer-Nucleic Acid Nanocomplexes Improves Nonviral Gene Transfer

    Science.gov (United States)

    Grigsby, Christopher L.; Ho, Yi-Ping; Lin, Chao; Engbersen, Johan F. J.; Leong, Kam W.

    2013-11-01

    As the designs of polymer systems used to deliver nucleic acids continue to evolve, it is becoming increasingly apparent that the basic bulk manufacturing techniques of the past will be insufficient to produce polymer-nucleic acid nanocomplexes that possess the uniformity, stability, and potency required for their successful clinical translation and widespread commercialization. Traditional bulk-prepared products are often physicochemically heterogeneous and may vary significantly from one batch to the next. Here we show that preparation of bioreducible nanocomplexes with an emulsion-based droplet microfluidic system produces significantly improved nanoparticles that are up to fifty percent smaller, more uniform, and are less prone to aggregation. The intracellular integrity of nanocomplexes prepared with this microfluidic method is significantly prolonged, as detected using a high-throughput flow cytometric quantum dot Förster resonance energy transfer nanosensor system. These physical attributes conspire to consistently enhance the delivery of both plasmid DNA and messenger RNA payloads in stem cells, primary cells, and human cell lines. Innovation in processing is necessary to move the field toward the broader clinical implementation of safe and effective nonviral nucleic acid therapeutics, and preparation with droplet microfluidics represents a step forward in addressing the critical barrier of robust and reproducible nanocomplex production.

  3. Cationic liposome-nucleic acid nanoparticle assemblies with applications in gene delivery and gene silencing.

    Science.gov (United States)

    Majzoub, Ramsey N; Ewert, Kai K; Safinya, Cyrus R

    2016-07-28

    Cationic liposomes (CLs) are synthetic carriers of nucleic acids in gene delivery and gene silencing therapeutics. The introduction will describe the structures of distinct liquid crystalline phases of CL-nucleic acid complexes, which were revealed in earlier synchrotron small-angle X-ray scattering experiments. When mixed with plasmid DNA, CLs containing lipids with distinct shapes spontaneously undergo topological transitions into self-assembled lamellar, inverse hexagonal, and hexagonal CL-DNA phases. CLs containing cubic phase lipids are observed to readily mix with short interfering RNA (siRNA) molecules creating double gyroid CL-siRNA phases for gene silencing. Custom synthesis of multivalent lipids and a range of novel polyethylene glycol (PEG)-lipids with attached targeting ligands and hydrolysable moieties have led to functionalized equilibrium nanoparticles (NPs) optimized for cell targeting, uptake or endosomal escape. Very recent experiments are described with surface-functionalized PEGylated CL-DNA NPs, including fluorescence microscopy colocalization with members of the Rab family of GTPases, which directly reveal interactions with cell membranes and NP pathways. In vitro optimization of CL-DNA and CL-siRNA NPs with relevant primary cancer cells is expected to impact nucleic acid therapeutics in vivoThis article is part of the themed issue 'Soft interfacial materials: from fundamentals to formulation'. PMID:27298431

  4. Design of polymer motifs for nucleic acid recognition and assembly stabilization

    Science.gov (United States)

    Zhou, Zhun

    This dissertation describes the synthesis and assembly of bio-functional polymers and the applications of these polymers to drug encapsulation, delivery, and multivalent biomimetic macromolecular recognition between synthetic polymer and nucleic acids. The main content is divided into three parts: (1) polyacidic domains as strongly stabilizing design elements for aqueous phase polyacrylate diblock assembly; (2) small molecule/polymer recognition triggered macromolecular assembly and drug encapsulation; (3) trizaine derivatized polymer as a novel class of "bifacial polymer nucleic acid" (bPoNA) and applications of bPoNA to nanoparticle loading of DNA/RNA, silencing delivery as well as control of aptamer function. Through the studies in part (1) and part (2), it was demonstrated that well-designed polymer motifs are not only able to enhance assemblies driven by non-specific hydrophobic effect, but are also able to direct assemblies based on specific recognitions. In part (3) of this dissertation, this concept was further extended by the design of polyacrylate polymers that are capable of discrete and robust hybridization with nucleic acids. This surprising finding demonstrated both fundamental and practical applications. Overall, these studies provided insights into the rational design elements for improving the bio-functions of synthetic polymers, and significantly expanded the scope of biological applications in which polymers synthesized via controlled radical polymerization may play a role.

  5. NaVirCept - Nucleic Acid-Based Anti-Viral Project

    International Nuclear Information System (INIS)

    Vaccines are generally considered to be the most effective countermeasures to bacterial and viral diseases, however, licensed vaccines against many disease agents are either not available or their efficacies have not been demonstrated. Vaccines are generally agent specific in terms of treatment spectrum and are subject to defeat through natural mutation or through directed efforts. With respect to viral therapeutics, one of the major limitations associated with antiviral drugs is acquired drug resistance caused by antigenic shift or drift. A number of next-generation prophylactic and/or therapeutic measures are on the horizon. Of these, nucleic acid-based drugs are showing great antiviral potential. These drugs elicit long-lasting, broad spectrum protective immune responses, especially to respiratory viral pathogens. The Nucleic Acid-Based Antiviral (NaVirCept) project provides the opportunity to demonstrate the effectiveness of novel medical countermeasures against military-significant endemic and other viral threat agents. This project expands existing DRDC drug delivery capability development, in the form of proprietary liposome intellectual property, by coupling it with leading-edge nucleic acid-based technology to deliver effective medical countermeasures that will protect deployed personnel and the warfighter against a spectrum of viral disease agents. The technology pathway will offer a means to combat emerging viral diseases or modified threat agents such as the bird flu or reconstructed Spanish flu without going down the laborious, time-consuming and expensive paths to develop countermeasures for each new and/or emerging viral disease organism.(author)

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

    Science.gov (United States)

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

    2015-11-11

    Programmable and algorithmic behaviors of DNA molecules allow one to control the structures of DNA-assembled materials with nanometer precision and to construct complex networks with digital and analog behaviors. Here we developed a way of integrating a DNA-strand-displacement circuit with self-assembly of spherical nucleic acids, wherein a single DNA strand was used to initiate and catalyze the operation of upstream circuits to release a single strand that subsequently triggers self-assembly of spherical nucleic acids in downstream circuits, realizing a programmable kinetic control of self-assembly of spherical nucleic acids. Through utilizing this method, single-nucleotide polymorphisms or indels occurring at different positions of a sequence of oligonucleotide were unambiguously discriminated. We provide here a sophisticated way of combining the DNA-strand-displacement-based characteristic of DNA with the distinct assembly properties of inorganic nanoparticles, which may find broad potential applications in the fabrication of a wide range of complex multicomponent devices and architectures. PMID:26485090

  7. Polypyrrole-polyvinyl sulphonate film based disposable nucleic acid biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Prabhakar, Nirmal [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi 110012 (India); Centre for Biomedical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi 110016 (India); Arora, Kavita [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi 110012 (India); Singh, Surinder P. [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi 110012 (India); Pandey, Manoj K. [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi 110012 (India); Singh, Harpal [Centre for Biomedical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi 110016 (India); Malhotra, Bansi D. [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi 110012 (India)]. E-mail: bansi.malhotra@gmail.com

    2007-04-18

    Double stranded calf thymus deoxyribonucleic acid entrapped polypyrrole-polyvinyl sulphonate (dsCT-DNA-PPy-PVS) films fabricated onto indium-tin-oxide (ITO) coated glass plates have been used to detect organophosphates such as chlorpyrifos and malathion. These disposable dsCT-DNA-PPy-PVS/ITO bioelectrodes have been characterized using cyclic voltammetry, Fourier-transform-infra-red (FTIR) spectroscopy and atomic force microscopy (AFM), respectively. These biosensing electrodes have a response time of 30 s, are stable for about 5 months when stored in desiccated conditions at 25 deg. C and can be used to amperometrically detect chlorpyrifos (0.0016-0.025 ppm) and malathion (0.17-5.0), respectively. The additive effect of these pesticides on the amperometric response of the disposable dsCT-DNA-PPy-PVS/ITO bioelectrodes has also been investigated.

  8. Stable carbon isotope analysis of nucleic acids to trace sources of dissolved substrates used by estuarine bacteria.

    OpenAIRE

    Coffin, R B; Velinsky, D J; R. Devereux; Price, W A; Cifuentes, L A

    1990-01-01

    The natural abundance of stable carbon isotopes measured in bacterial nucleic acids extracted from estuarine bacterial concentrates was used to trace sources of organic matter for bacteria in aquatic environments. The stable carbon isotope ratios of Pseudomonas aeruginosa and nucleic acids extracted from cultures resembled those of the carbon source on which bacteria were grown. The carbon isotope discrimination between the substrate and total cell carbon from bacterial cultures averaged 2.3%...

  9. Efficacy of Nucleic Acid Probes for Detection of Poliovirus in Water Disinfected by Chlorine, Chlorine Dioxide, Ozone, and UV Radiation

    OpenAIRE

    Moore, Norman J.; Margolin, Aaron B.

    1994-01-01

    MilliQ water was inoculated with poliovirus type 1 strain LSc-1 and was treated with disinfectants, including chlorine, chlorine dioxide, ozone, and UV light. No relationship between probes and plaque assays were seen, demonstrating that viral nucleic acids were not destroyed. These findings suggest that nucleic acid probes cannot distinguish between infectious and noninfectious viruses and cannot be used in the evaluation of treated waters.

  10. Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples ▿ †

    OpenAIRE

    Hata, Akihiko; Katayama, Hiroyuki; Kitajima, Masaaki; Visvanathan, Chettiyappan; Nol, Chea; Furumai, Hiroaki

    2011-01-01

    Inhibitors that reduce viral nucleic acid extraction efficiency and interfere with cDNA synthesis and/or polymerase activity affect the molecular detection of viruses in aquatic environments. To overcome these significant problems, we developed a methodology for assessing nucleic acid yields and DNA amplification efficiencies for environmental water samples. This involved adding particles of adenovirus type 5 and murine norovirus and newly developed primer-sharing controls, which are amplifie...

  11. MNAzymes, a Versatile New Class of Nucleic Acid Enzymes That Can Function as Biosensors and Molecular Switches

    OpenAIRE

    Mokany, Elisa; Bone, Simon M.; Young, Paul E; Doan, Tram B.; Todd, Alison V.

    2009-01-01

    To increase the versatility and utility of nucleic acid enzymes, we developed multicomponent complexes, known as MNAzymes, which produce amplified “output” signals in response to specific “input” signals. Multiple oligonucleotide partzymes assemble into active MNAzymes only in the presence of an input assembly facilitator such as a target nucleic acid. Once formed, MNAzymes catalytically modify a generic substrate, generating an amplified output signal that heralds the presence of the target ...

  12. A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories

    OpenAIRE

    Lou, Jerry J.; Mirsadraei, Leili; Sanchez, Desiree E.; Wilson, Ryan W.; Shabihkhani, Maryam; Gregory M Lucey; Wei, Bowen; Singer, Elyse J.; Mareninov, Sergey; Yong, William H.

    2013-01-01

    Frozen biospecimens are crucial for translational research and contain well preserved nucleic acids and protein. However, the risk for catastrophic freezer failure as well as space, cost, and environmental concerns argue for evaluating long-term room temperature storage alternatives. Formalin-fixed paraffin embedded (FFPE) tissues have great value but their use is limited by cross-linking and fragmentation of nucleic acids, as well as loss of enzymatic activity. Stabilization solutions can no...

  13. Evolution of the deaminase fold and multiple origins of eukaryotic editing and mutagenic nucleic acid deaminases from bacterial toxin systems

    OpenAIRE

    Iyer, Lakshminarayan M; Zhang, Dapeng; Rogozin, Igor B.; Aravind, L.

    2011-01-01

    The deaminase-like fold includes, in addition to nucleic acid/nucleotide deaminases, several catalytic domains such as the JAB domain, and others involved in nucleotide and ADP-ribose metabolism. Using sensitive sequence and structural comparison methods, we develop a comprehensive natural classification of the deaminase-like fold and show that its ancestral version was likely to operate on nucleotides or nucleic acids. Consequently, we present evidence that a specific group of JAB domains ar...

  14. The interaction of poly(ethylenimine) with nucleic acids and its use in determination of nucleic acids based on light scattering

    Science.gov (United States)

    Zhou, Ying-lin; Li, Yuan-zong

    2004-01-01

    For the first time, poly(ethylenimine) (PEI) was used to determine nucleic acids with a light scattering technique using a common spectrofluorometer. The interaction of PEI with DNA results in greatly enhanced intensity of light scattering at 300 nm, which is caused by the formation of the big particles between DNA and PEI. Based on this, a new quantitative method for nucleic acid determination in aqueous solutions has been developed. Under the optimum conditions, the enhanced intensity of light scattering is proportional to the concentration of nucleic acid in the range of 0.01-10.0 μg ml -1 for herring sperm DNA (hsDNA), 0.02-10.0 μg ml -1 for calf thymus DNA (ctDNA), 0.02-20.0 μg ml -1 for yeast RNA (yRNA). The detection limits are 5.3, 9.9, and 13.7 ng ml -1, respectively. Synthetic samples were determined satisfactorily. At the same time, the light scattering technique has been successfully used to obtain the information on the effects of pH and ionic strength on the formation and the stability of the DNA/PEI complex, which is important in some fields such as genetic engineering and gene transfer. Using ethidium bromide (EB) as a fluorescent probe, the binding of PEI with hsDNA was studied. Both the binding constant of EB with DNA and the number of binding sites per nucleotide decrease with increasing concentration of PEI, indicating noncompetitive inhibition of EB binding to DNA in the presence of PEI. And the association constant of PEI to DNA obtained is 1.2×10 5 M -1. IR-spectra show that PEI interacts with DNA through both the phosphate groups and the bases of DNA and the formation of DNA/PEI complex may cause the change of the conformation of the DNA secondary structure, which is also proved by UV-spectra.

  15. A novel automated device for rapid nucleic acid extraction utilizing a zigzag motion of magnetic silica beads.

    Science.gov (United States)

    Yamaguchi, Akemi; Matsuda, Kazuyuki; Uehara, Masayuki; Honda, Takayuki; Saito, Yasunori

    2016-02-01

    We report a novel automated device for nucleic acid extraction, which consists of a mechanical control system and a disposable cassette. The cassette is composed of a bottle, a capillary tube, and a chamber. After sample injection in the bottle, the sample is lysed, and nucleic acids are adsorbed on the surface of magnetic silica beads. These magnetic beads are transported and are vibrated through the washing reagents in the capillary tube under the control of the mechanical control system, and thus, the nucleic acid is purified without centrifugation. The purified nucleic acid is automatically extracted in 3 min for the polymerase chain reaction (PCR). The nucleic acid extraction is dependent on the transport speed and the vibration frequency of the magnetic beads, and optimizing these two parameters provided better PCR efficiency than the conventional manual procedure. There was no difference between the detection limits of our novel device and that of the conventional manual procedure. We have already developed the droplet-PCR machine, which can amplify and detect specific nucleic acids rapidly and automatically. Connecting the droplet-PCR machine to our novel automated extraction device enables PCR analysis within 15 min, and this system can be made available as a point-of-care testing in clinics as well as general hospitals. PMID:26772121

  16. 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.

  17. How does hydroxyl introduction influence the double helical structure: the stabilization of an altritol nucleic acid:ribonucleic acid duplex

    Czech Academy of Sciences Publication Activity Database

    Ovaere, M.; Šponer, Jiří; Šponer, Judit E.; Herdewijn, P.; Van Meervelt, L.

    2012-01-01

    Roč. 40, č. 15 (2012), s. 7573-7583. ISSN 0305-1048 R&D Projects: GA ČR(CZ) GAP208/11/1822; GA ČR(CZ) GAP208/10/2302; GA ČR(CZ) GA203/09/1476 Institutional research plan: CEZ:AV0Z50040702 Keywords : duplex * atritol nucleic acids * O2' and O4' hydroxyl group Subject RIV: BO - Biophysics Impact factor: 8.278, year: 2012

  18. A four-year clinical evaluation of acid etched bridges

    International Nuclear Information System (INIS)

    Thirty six (36) three unit acid etched bridges (20 posterior and 16 anterior) were clinically evaluated in relation to retention, cracking in the porcelain and caries status of the abutment teeth. The results showed that after 4 years 26 bridges (72.2 %) were successfully retained, one porcalin facing had fractured and none of the abutments showed evidence of caries during the period of the study. (author)

  19. Nucleic Acid Encoding A Lectin-Derived Progenitor Cell Preservation Factor

    Science.gov (United States)

    Colucci, M. Gabriella; Chrispeels, Maarten J.; Moore, Jeffrey G.

    2001-10-30

    The invention relates to an isolated nucleic acid molecule that encodes a protein that is effective to preserve progenitor cells, such as hematopoietic progenitor cells. The nucleic acid comprises a sequence defined by SEQ ID NO:1, a homolog thereof, or a fragment thereof. The encoded protein has an amino acid sequence that comprises a sequence defined by SEQ ID NO:2, a homolog thereof, or a fragment thereof that contains an amino acid sequence TNNVLQVT. Methods of using the encoded protein for preserving progenitor cells in vitro, ex vivo, and in vivo are also described. The invention, therefore, include methods such as myeloablation therapies for cancer treatment wherein myeloid reconstitution is facilitated by means of the specified protein. Other therapeutic utilities are also enabled through the invention, for example, expanding progenitor cell populations ex vivo to increase chances of engraftation, improving conditions for transporting and storing progenitor cells, and facilitating gene therapy to treat and cure a broad range of life-threatening hematologic diseases.

  20. Stable dye-labelled oligonucleotide-nanoparticle conjugates for nucleic acid detection

    Science.gov (United States)

    Barrett, Lee; Dougan, Jennifer A.; Faulds, Karen; Graham, Duncan

    2011-08-01

    Metallic nanoparticles functionalized with oligonucleotides are used for a number of nucleic acid detection strategies. However, oligonucleotide-nanoparticle conjugates suffer from a lack of stability when exposed to certain conditions associated with DNA detection assays. In this study, we report the synthesis of thiol and thioctic acid-modified oligonucleotide gold nanoparticle (OGNs) conjugates functionalized with a dye label and varying spacer groups. The thioctic acid-modified conjugates exhibit increased stability when treated with dithiothreitol (DTT) compared to the more commonly used thiol modification. When the dye labelled oligonucleotide nanoparticle conjugates are exposed to the same conditions there is a pronounced increase in the stability for both thioctic acid and thiol modified sequences. These results open up the possibility of simply using a dye label to enhance the stability of oligonucleotide-nanoparticle conjugates in DNA detection assays where the enhanced stability of the conjugate system can be advantageous in more complex biological environments.

  1. Solar-thermal complex sample processing for nucleic acid based diagnostics in limited resource settings.

    Science.gov (United States)

    Gumus, Abdurrahman; Ahsan, Syed; Dogan, Belgin; Jiang, Li; Snodgrass, Ryan; Gardner, Andrea; Lu, Zhengda; Simpson, Kenneth; Erickson, David

    2016-05-01

    The use of point-of-care (POC) devices in limited resource settings where access to commonly used infrastructure, such as water and electricity, can be restricted represents simultaneously one of the best application fits for POC systems as well as one of the most challenging places to deploy them. Of the many challenges involved in these systems, the preparation and processing of complex samples like stool, vomit, and biopsies are particularly difficult due to the high number and varied nature of mechanical and chemical interferents present in the sample. Previously we have demonstrated the ability to use solar-thermal energy to perform PCR based nucleic acid amplifications. In this work demonstrate how the technique, using similar infrastructure, can also be used to perform solar-thermal based sample processing system for extracting and isolating Vibrio Cholerae nucleic acids from fecal samples. The use of opto-thermal energy enables the use of sunlight to drive thermal lysing reactions in large volumes without the need for external electrical power. Using the system demonstrate the ability to reach a 95°C threshold in less than 5 minutes and maintain a stable sample temperature of +/- 2°C following the ramp up. The system is demonstrated to provide linear results between 10(4) and 10(8) CFU/mL when the released nucleic acids were quantified via traditional means. Additionally, we couple the sample processing unit with our previously demonstrated solar-thermal PCR and tablet based detection system to demonstrate very low power sample-in-answer-out detection. PMID:27231636

  2. TMPyP4, a Stabilizer of Nucleic Acid Secondary Structure, Is a Novel Acetylcholinesterase Inhibitor.

    Directory of Open Access Journals (Sweden)

    Nana Fujiwara

    Full Text Available The porphyrin compound, TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridylporphine, is widely used as a photosensitizer and a modulator of nucleic acid secondary structure stability. Our group recently showed in cultured cells and forebrain slice cultures that this compound can also down regulate expression of Tyrosine hydroxylase (Th, which encodes the rate-limiting enzyme in catecholamine biosynthesis, by stabilizing DNA secondary structures in the Th proximal promoter. The current study sought to establish whether treatment with TMPyP4 could modify mouse Th expression levels in vivo. Intraperitoneal administration of low TMPyP4 doses (10mg/kg, similar to those used for photosensitization, did not significantly reduce Th transcript levels in several catecholaminergic regions. Administration of a high dose (40 mg/kg, similar to those used for tumor xenograph reduction, unexpectedly induced flaccid paralysis in an age and sex-dependent manner. In vitro analyses revealed that TMPyP4, but not putative metabolites, inhibited Acetylcholinesterase activity and pre-treatment of TMPyP4 with Hemeoxygenase-2 (HO-2 rescued Acetylcholinesterase function. Age-dependent differences in HO-2 expression levels may account for some of the variable in vivo effects of high TMPyP4 doses. Together, these studies indicate that only low doses of TMPyP4, such as those typically used for photosensitization, are well tolerated in vivo. Thus, despite its widespread use in vitro, TMPyP4 is not ideal for modifying neuronal gene expression in vivo by manipulating nucleic acid secondary structure stability, which highlights the need to identify more clinically suitable compounds that can modulate nucleic acid secondary structure and gene expression.

  3. CJD and Scrapie Require Agent-Associated Nucleic Acids for Infection.

    Science.gov (United States)

    Botsios, Sotirios; Manuelidis, Laura

    2016-08-01

    Unlike Alzheimer's and most other neurodegenerative diseases, Transmissible Spongiform Encephalopathies (TSEs) are all caused by actively replicating infectious particles of viral size and density. Different strain-specific TSE agents cause CJD, kuru, scrapie and BSE, and all behave as latent viruses that evade adaptive immune responses and can persist for years in lymphoreticular tissues. A foreign viral structure with a nucleic acid genome best explains these TSE strains and their endemic and epidemic spread in susceptible species. Nevertheless, it is widely believed that host prion protein (PrP), without any genetic material, encodes all these strains. We developed rapid infectivity assays that allowed us to reproducibly isolate infectious particles where >85% of the starting titer separated from the majority of host components, including PrP. Remarkably, digestion of all forms of PrP did not reduce brain particle titers. To ask if TSE agents, as other viruses, require nucleic acids, we exposed high titer FU-CJD and 22L scrapie particles to potent nucleases. Both agent-strains were propagated in GT1 neuronal cells to avoid interference by complex degenerative brain changes that can impede nuclease digestions. After exposure to nucleases that are active in sarkosyl, infectivity of both agents was reproducibly reduced by ≥99%. No gold-stained host proteins or any form of PrP were visibly altered by these nucleases. In contrast, co-purifying protected mitochondrial DNA and circular SPHINX DNAs were destroyed. These findings demonstrate that TSE agents require protected genetic material to infect their hosts, and should reopen investigation of essential agent nucleic acids. J. Cell. Biochem. 117: 1947-1958, 2016. © 2016 Wiley Periodicals, Inc. PMID:26773845

  4. Solar-thermal complex sample processing for nucleic acid based diagnostics in limited resource settings

    Science.gov (United States)

    Gumus, Abdurrahman; Ahsan, Syed; Dogan, Belgin; Jiang, Li; Snodgrass, Ryan; Gardner, Andrea; Lu, Zhengda; Simpson, Kenneth; Erickson, David

    2016-01-01

    The use of point-of-care (POC) devices in limited resource settings where access to commonly used infrastructure, such as water and electricity, can be restricted represents simultaneously one of the best application fits for POC systems as well as one of the most challenging places to deploy them. Of the many challenges involved in these systems, the preparation and processing of complex samples like stool, vomit, and biopsies are particularly difficult due to the high number and varied nature of mechanical and chemical interferents present in the sample. Previously we have demonstrated the ability to use solar-thermal energy to perform PCR based nucleic acid amplifications. In this work demonstrate how the technique, using similar infrastructure, can also be used to perform solar-thermal based sample processing system for extracting and isolating Vibrio Cholerae nucleic acids from fecal samples. The use of opto-thermal energy enables the use of sunlight to drive thermal lysing reactions in large volumes without the need for external electrical power. Using the system demonstrate the ability to reach a 95°C threshold in less than 5 minutes and maintain a stable sample temperature of +/− 2°C following the ramp up. The system is demonstrated to provide linear results between 104 and 108 CFU/mL when the released nucleic acids were quantified via traditional means. Additionally, we couple the sample processing unit with our previously demonstrated solar-thermal PCR and tablet based detection system to demonstrate very low power sample-in-answer-out detection. PMID:27231636

  5. Improved Experimental Techniques for Analyzing Nucleic Acid Transport Through Protein Nanopores in Planar Lipid Bilayers

    Science.gov (United States)

    Costa, Justin A.

    The translocation of nucleic acid polymers across cell membranes is a fundamental requirement for complex life and has greatly contributed to genomic molecular evolution. The diversity of pathways that have evolved to transport DNA and RNA across membranes include protein receptors, active and passive transporters, endocytic and pinocytic processes, and various types of nucleic acid conducting channels known as nanopores. We have developed a series of experimental techniques, collectively known as "Wicking", that greatly improves the biophysical analysis of nucleic acid transport through protein nanopores in planar lipid bilayers. We have verified the Wicking method using numerous types of classical ion channels including the well-studied chloride selective channel, CLIC1. We used the Wicking technique to reconstitute α-hemolysin and found that DNA translocation events of types A and B could be routinely observed using this method. Furthermore, measurable differences were observed in the duration of blockade events as DNA length and composition was varied, consistent with previous reports. Finally, we tested the ability of the Wicking technology to reconstitute the dsRNA transporter Sid-1. Exposure to dsRNAs of increasing length and complexity showed measurable differences in the current transitions suggesting that the charge carrier was dsRNA. However, the translocation events occurred so infrequently that a meaningful electrophysiological analysis was not possible. Alterations in the lipid composition of the bilayer had a minor effect on the frequency of translocation events but not to such a degree as to permit rigorous statistical analysis. We conclude that in many instances the Wicking method is a significant improvement to the lipid bilayer technique, but is not an optimal method for analyzing transport through Sid-1. Further refinements to the Wicking method might have future applications in high throughput DNA sequencing, DNA computation, and molecular

  6. 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/). PMID:24316579

  7. Terbium fluorescence as a sensitive, inexpensive probe for UV-induced damage in nucleic acids

    International Nuclear Information System (INIS)

    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) (Tb3+). Single-stranded oligonucleotides greatly enhance the Tb3+ emission, but duplex DNA does not. With the use of a DNA hairpin probe complementary to the oligonucleotide of interest, the Tb3+/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 Tb3+, producing a detectable signal directly proportional to the amount of DNA damage. This allows the Tb3+/hairpin probe to be used for sensitive quantification of UV-induced DNA damage. The Tb3+/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

  8. Cell-free nucleic acids as a non-invasive route for investigating atherosclerosis.

    Science.gov (United States)

    Cerne, Darko; Bajalo, Jana Lukac

    2014-01-01

    Metabolic syndrome is directly linked with atherosclerotic burden and cell-free nucleic acids (cf-NA) analysis has recently emerged as a novel research tool in atherosclerosis practice and research. cf-NA are nucleic acids (DNA, mRNA, miRNA, mitochondrial DNA) found in plasma and cell-free fractions of various other biological fluids. They have all the characteristics of the nucleic acids in the cells of their origin, thus constituting an emerging field for non-invasive assessment. Initially, quantitative and qualitative analysis of cf-NA has been accepted as clinically useful in non-invasive prenatal diagnosis, and in the diagnosis and monitoring of numerous cancers. As to atherosclerosis, cf-NA analysis poses an important challenge in diagnosis and prognostic evaluation of acute coronary syndrome, in prediction of cardiovascular disease, in non-invasive early detection of atherosclerosis and understanding its pathological mechanism in vivo, in assessing various issues of treatment for atherosclerosis in vivo, and in the unique simultaneous measurement of mRNA levels and protein concentrations in a single sample of plasma. Examples of its use are presented in this review. Besides the advances in technologies, the precise evaluation and optimization of pre-analytical and analytical aspects of cf-NA analysis have impacted importantly on the reliability of test results. We have, therefore, reviewed the most important analytical considerations. Further clinical studies and analytical improvements will answer the question as to whether cf-NA, as novel biomarkers, can be reliably applied clinically in non-invasive, early diagnosis and monitoring of the vulnerable atherosclerotic plaques of patients who could suffer from acute coronary syndrome. PMID:24320033

  9. Stabilizing Role of Metallophilic HgII ... HgII Interactions in Nucleic Acids

    Czech Academy of Sciences Publication Activity Database

    Benda, Ladislav; Tanaka, Y.; Sychrovský, Vladimír; Straka, Michal

    Praha: -, 2011. s. 35-35. [Quantum Bioinorganic Chemistry Conference /3./. 25.06.2011-28.06.2011, Český Krumlov] R&D Projects: GA ČR GA203/09/2037; GA ČR GAP205/10/0228 Grant ostatní: European Reintegration Grant(XE) 230955 Institutional research plan: CEZ:AV0Z40550506 Keywords : metallophilic HgII ... HgII interactions * mispairing nucleic acids * metallophilic interactions Subject RIV: CF - Physical ; Theoretical Chemistry

  10. 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...

  11. Intrinsic Nucleic Acid Dynamics Modulates HIV-1 Nucleocapsid Protein Binding to Its Targets

    OpenAIRE

    Bazzi, Ali; Zargarian, Loussiné; Chaminade, Françoise; De Rocquigny, Hugues; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2012-01-01

    HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem. To further u...

  12. Use of molecular nuclear methods in communicable diseases: From nucleic acid hybridization to proteomics

    International Nuclear Information System (INIS)

    In spite of decades of intensive research on communicable diseases, infection agents still remain a major cause of morbidity and mortality in humans. These rates have driven a major resurgence in biological research efforts for the identification of new targets for vaccine development, drugs and diagnostics assays products. Molecular nuclear techniques focused in nucleic acid hybridization and DNA amplification and more recently proteomic approaches are employed to address questions related to communicable diseases. In nucleic acid hybridization, denatured DNA or RNA is immobilized on an inert support, so that bound sequences are available for hybridization with an added nucleic acid radioactive probe to facilitate analysis. After extensive washing, hybrids are detected by autoradiography or phosphoimager analyzer. Polymerase chain reaction (PCR), is a very sensitive method for nucleic acid amplification used when the targets are scarce in the cells or in clinical material. Differently, the challenge of proteome analysis lies with the task of achieving a combination of high-throughput screening while maintaining high sensitivity for the detection of low copy number proteins. Many different detection technologies have been developed to improve visualization of proteins in protein analysis and radiolabelling is one of the strategies for protein detection. For metabolic experiments, proteins must be labelled with an appropriate radioactive isotope in vivo prior to isolation by electrophoretic analysis. Additionally, protein phosphorylation, an important post-translational modification, is analyzed by using 32P for in vivo labelling of phosphorylated proteins and further identification by mass spectrometry. The major problems which limit proteomic studies deal with the comparative analysis of 2DE gel images. In conventional methodology, the protein samples to be compared are separated independently on different gels. Using a 2DE image analysis software spots are

  13. Restoration of nucleic acid biosynthesis after clinical death and factors stimulating the process in vivo.

    Science.gov (United States)

    Konikova, A S; Petukhova, L M; Pogossova, A V; Vinarskaya, A A; Nikulin, V I

    1975-01-01

    The biosynthesis of RNA and DNA falls almost to zero in 60 min after the death of rabbits from anoxia, in all the organs of the body. Rapid artificial cooling of the rabbits to 20 degrees C undertaken within 10 min after death preserved nucleic acid biosynthesis and permitted restoration of life 3-4 h after death, with recovery beginning in 60 min. During the reanimation the addition of ATP to the blood stimulated the restoration of RNA biosynthesis in the spinal cord to a considerable extent; the addition of cocarboxylase to the blood promoted cardiac RNA biosynthesis as well as cardiac and pancreatic DNA biosynthesis during recovery. PMID:1197938

  14. Peptide nucleic acid-anthraquinone conjugates: strand invasion and photoinduced cleavage of duplex DNA.

    OpenAIRE

    Armitage, B.; T. Koch; Frydenlund, H; Orum, H; Batz, H G; Schuster, G B

    1997-01-01

    A bis-peptide nucleic acid (PNA)-anthraquinone imide (AQI) conjugate has been synthesized and shown to form strand invasion complexes with a duplex DNA target. The two arms of the bis-PNA each consist of five consecutive thymine residues and are linked by a flexible, hydrophilic spacer. Probing with potassium permanganate reveals that the bis-PNA complexes to duplex DNA at A5.T5sites with local displacement of the T5DNA strand. The 5 bp sequence targeted by the PNA is the shortest strand inva...

  15. A Single-Tube Nucleic Acid Extraction, Amplification, and Detection Method Using Aluminum Oxide

    OpenAIRE

    Dames, Shale; Bromley, L. Kathryn; Herrmann, Mark; Elgort, Marc; Erali, Maria; Smith, Roger; Voelkerding, Karl V.

    2006-01-01

    A disposable 0.2-ml polymerase chain reaction (PCR) tube modified with an aluminum oxide membrane (AOM) has been developed for the extraction, amplification, and detection of nucleic acids. To assess the dynamic range of AOM tubes for real-time PCR, quantified herpes simplex virus (HSV) DNA was used to compare AOM tubes to standard PCR tubes. AOM PCR tubes used for amplification and detection of quantified HSV-1 displayed a crossing threshold (CT) shift 0.1 cycles greater than PCR tube contro...

  16. 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

    Due to the extensive use of nucleic acid and protein analysis of bacterial samples, there is a need for simple and rapid extraction protocols for both plasmid DNA and RNA molecules as well as reporter proteins like the green fluorescent protein (GFP). In this report, an electropermeability...... can be avoided and the transiently formed pores can be closed again and the cells survive. This method has been used to extract RNA and GFP molecules under conditions of electropermeability. Plasmid DNA could be recovered when the applied voltage was increased to 2 V, thus causing complete cell lysis....

  17. Optimal design of parallel triplex forming oligonucleotides containing Twisted Intercalating Nucleic Acids--TINA

    DEFF Research Database (Denmark)

    Schneider, Uffe V; Mikkelsen, Nikolaj D; Jøhnk, Nina;

    2010-01-01

    Twisted intercalating nucleic acid (TINA) is a novel intercalator and stabilizer of Hoogsteen type parallel triplex formations (PT). Specific design rules for position of TINA in triplex forming oligonucleotides (TFOs) have not previously been presented. We describe a complete collection of easy...... on PT is remarkably high (between 7.4 and 15.2 degrees C) compared to antiparallel duplexes (between 3.8 and 9.4 degrees C). The specificity of PT by Delta T(m) increases when shorter TFOs and higher pH are chosen. To increase Delta Tms, base mismatches should be placed in the center of the TFO and...

  18. Single and double stranded DNA detection using locked nucleic acid (LNA) functionalized nanoparticles

    Science.gov (United States)

    McKenzie, Fiona; Stokes, Robert; Faulds, Karen; Graham, Duncan

    2008-08-01

    Gold and silver nanoparticles functionalized with oligonucleotides can be used for the detection of specific sequences of DNA. We show that gold nanoparticles modified with locked nucleic acid (LNA) form stronger duplexes with a single stranded DNA target and offer better discrimination against single base pair mismatches than analogous DNA probes. Our LNA nanoparticle probes have also been used to detect double stranded DNA through triplex formation, whilst still maintaining selectivity for only complementary targets. Nanoparticle conjugates embedded with suitable surface enhanced resonance Raman scattering (SERRS) labels have been synthesized enabling simultaneous detection and identification of multiple DNA targets.

  19. Action of polyvinylsulphate on metabolism of nucleic acids affected by radiation

    International Nuclear Information System (INIS)

    Polyvinyl sulphate (PVS) as a nuclease inhibitor was used to protect endogenic nucleic acids from irradiation-activated nucleases. The survival of 700 rad irradiated rats was 33% and 60% in the control and PVS-treated (25 mg/kg) groups, respectively. PVS intravenously injected to 1000 R irradiated animals at 50 mg/kg inhibited DNase I in the liver and spleen and stimulated DNase 2 in the liver. A partial restoration of DNA level in the liver was observed simultaneously with the inhibitory effect on alkaline nucleases (no restoration effect had been found in the spleen)

  20. Colostral transmission of bluetongue virus nucleic acid among newborn dairy calves in California.

    Science.gov (United States)

    Mayo, C E; Crossley, B M; Hietala, S K; Gardner, I A; Breitmeyer, R E; Maclachlan, N James

    2010-08-01

    There have been substantial recent changes in the global distribution and nature of bluetongue virus (BTV) infection of ungulates, perhaps as a result of climate change. To evaluate the epidemiology of BTV infection in California, an area historically endemic for the virus, we monitored newborn dairy calves at different sites for 1 year for the presence of BTV RNA and virus-specific antibodies. The data confirm both localized, vector-mediated, seasonal transmission of BTV as well as dissemination of BTV and/or viral nucleic acid to newborn calves following ingestion of colostrum. PMID:20557494

  1. Convenient and scalable synthesis of fmoc-protected Peptide nucleic Acid backbone.

    Science.gov (United States)

    Feagin, Trevor A; Shah, Nirmal I; Heemstra, Jennifer M

    2012-01-01

    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. PMID:22848796

  2. Convenient and Scalable Synthesis of Fmoc-Protected Peptide Nucleic Acid Backbone

    OpenAIRE

    Feagin, Trevor A.; Shah, Nirmal I.; Heemstra, Jennifer M.

    2012-01-01

    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 mo...

  3. 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.

  4. The orientation of binding of human immunodeficiency virus reverse transcriptase on nucleic acid hybrids.

    OpenAIRE

    DeStefano, J J

    1995-01-01

    The binding of HIV reverse transcriptase (RT) to heteroduplexes was examined using a substrate consisting of a 42 nt chimeric nucleic acid composed. (5'-->3') of 23 nt of RNA and 19 of DNA. This chimera was hybridized to an internal region of a relatively long complementary DNA or RNA. When the chimera was bound to DNA and conditions limiting cleavage to a single binding event between the enzyme and substrate were employed initial RNase H-directed cleavages occurred 19-21 nt from the chimera ...

  5. Question 1: Peptide Nucleic Acids and the Origin and Homochirality of Life

    Science.gov (United States)

    Nielsen, Peter E.

    2007-10-01

    The possibilities of pseudo peptide DNA mimics like PNA (peptide nucleic acid) having a role for the prebiotic origin of life prior to an RNA world is discussed. In particular a scenario is proposed in which protocells with an achiral genetic material through several generations stepwise is converted into a chiral genetic material, e.g., by incorporation of RNA units. Provided that a sufficiently large sequence space is occupied, a selection process based on catalytic function in which a single cell (first common ancestor) has a definite evolutionary advantage, selection of this cell would by contingency also lock it into homochirality.

  6. Application of CCT Technique to Simulate Vibrational Spectra of Nucleic Acid Oligomers

    Czech Academy of Sciences Publication Activity Database

    Andrushchenko, Valery; Bouř, Petr

    Lviv : -, 2011. s. 41-41. [Methods and Applications of Computational Chemistry. International Symposium /4./. 28.06.2011-02.07.2011, Lviv] R&D Projects: GA ČR GAP208/10/0559; GA ČR GAP208/11/0105 Grant ostatní: AV ČR(CZ) M200550902 Institutional research plan: CEZ:AV0Z40550506 Keywords : IR spectroscopy * VCD spectroscopy * quantum chemistry spectra simulations * Cartesian coordinate transfer (CCT) * nucleic acids Subject RIV: CF - Physical ; Theoretical Chemistry

  7. 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.

  8. [Rapidly labelled low molecular weight components in nucleic acid preparations from plant cells].

    Science.gov (United States)

    Richter, G; Grotha, R

    1974-09-01

    After pulse-labelling with [(3)H]nucleosides and [(3)H]orotic acid of freely suspended callus cells of Petroselinum sativum and tissue fragments of the liverwort Riella helicophylla, rapidly labelled low molecular weight components were detected among the total nucleic acids when these were extracted in the presence of Mg(2+) and finally precipitated with alcohol. These highly labelled species could clearly be distinguished from the 5 S- and 4 S-RNA on the basis of their migration in agarose-polyacrylamide gels (2.4%) and their elution from Sephadex G-150 columns. No degradation was obtained with DNase and RNase. By using [(14)C]ATP as a marker it was found that the low molecular components consisted mainly of nucleoside triphosphates. Only small amounts of nucleoside diphosphates were detected, which were obviously formed by degradation of the former. Nucleic acid preparations free of nucleoside phosphates were obtained by using Mg-free extraction buffers containing EDTA. PMID:24458196

  9. 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.

  10. Sensitive, Efficient Quantitation of 13C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry for Applications in Stable Isotope Probing

    OpenAIRE

    Wilhelm, Roland; Szeitz, András; Klassen, Tara L; Mohn, William W.

    2014-01-01

    Stable isotope probing (SIP) of nucleic acids is a powerful tool for studying the functional traits of microbial populations within complex communities, but SIP involves a number of technical challenges. Many of the difficulties in DNA-SIP and RNA-SIP experiments can be effectively overcome with an efficient, sensitive method for quantitating the isotopic enrichment of nucleic acids. Here, we present a sensitive method for quantitating 13C enrichment of nucleic acids, requiring a few nanogram...

  11. Standard in vitro assays for protein-nucleic acid interactions--gel shift assays for RNA and DNA binding.

    Science.gov (United States)

    Mitchell, Sarah F; Lorsch, Jon R

    2014-01-01

    The characterization of protein-nucleic acid interactions is necessary for the study of a wide variety of biological processes. One straightforward and widely used approach to this problem is the electrophoretic mobility shift assay (EMSA), in which the binding of a nucleic acid to one or more proteins changes its mobility through a nondenaturing gel matrix. Usually, the mobility of the nucleic acid is reduced, but examples of increased mobility do exist. This type of assay can be used to investigate the affinity of the interaction between the protein and nucleic acid, the specificity of the interaction, the minimal binding site, and the kinetics of the interaction. One particular advantage of EMSA is the ability to analyze multiple proteins, or protein complexes, binding to nucleic acids. This assay is relatively quick and easy and utilizes equipment available in most laboratories; however, there are many variables that can only be determined empirically; therefore, optimization is necessary and can be highly dependent upon the system. The protocol described here is for the poly(A)-binding protein (PABP) binding to an unstructured RNA probe of 43 bases. While this may be a useful protocol for some additional assays, it is recommended that both reaction conditions and gel running conditions be tailored to the individual interaction to be probed. PMID:24674072

  12. Downregulation of p21(WAF1/CIP1) and estrogen receptor alpha in MCF-7 cells by antisense oligonucleotides containing locked nucleic acid (LNA)

    DEFF Research Database (Denmark)

    Jepsen, Jan Stenvang; Pfundheller, Henrik M; Lykkesfeldt, Anne E; Stenvang, Jan

    2004-01-01

    Locked nucleic acid (LNA) is a nucleic acid analog with very high affinity to complementary RNA and a promising compound in the field of antisense research. The intracellular localization and quantitative uptake of oligonucleotides containing LNA were found to be equivalent to those of phosphorot......Locked nucleic acid (LNA) is a nucleic acid analog with very high affinity to complementary RNA and a promising compound in the field of antisense research. The intracellular localization and quantitative uptake of oligonucleotides containing LNA were found to be equivalent to those of...

  13. Studies on the antibacterial and nucleic acid degradation property of Cassia alata

    Directory of Open Access Journals (Sweden)

    Elangomathavan Ramaraj

    2014-03-01

    Full Text Available This study aimed to screen preliminary phytochemical profile of the leaf extract of Cassia alata (L,one of the ethnomedicinal plants used by the Kani tribes of the Western Ghats, Tamilnadu, India. To evaluate the antimicrobial potential of multiple solvent based crude leaf extracts of the selected plant species C. alataagainst the few target human pathogenic microorganisms.Multiple solvents were used for the extraction of allelochemicals from thedried leaf material. Crudeextracts were subjected to evaluate their efficacy in controlling the target microorganism’s growth at in vitrolevel. Variation in the total nucleic acid content was also assessed in both control and treated bacterial samples. C. alata leaf extract chemical profile revealed the presence of secondary metabolites like tannins, phlobatanins, saponins, flavonoids, steroids, terpenoids and alkaloids. Crude extracts prepared in the Soxhlet apparatus ceased the growth of Salmonella typhi andPseudomonas aeruginosa. Proteus vulgaris andKlebsiella pneumoniaewere resistant even while fortified with 3 mg/ml extract concentration. Extract treated bacteria lack the total nucleic acid content, which proves and supports the presence of antimicrobial potential. Multiple solvent based C. alata leaf crude extracts have the potential to control one ofthe most important human pathogens S. typhi. This plant could be exploited further for the analysis of active principles towards the optimization of drug, as an evidence for sustainable utilization of the traditional knowledge on medicinal plants used by the Kani tribes herbal healers.

  14. Tailoring Conformation-Induced Chromism of Polythiophene Copolymers for Nucleic Acid Assay at Resource Limited Settings.

    Science.gov (United States)

    Rajwar, Deepa; Ammanath, Gopal; Cheema, Jamal Ahmed; Palaniappan, Alagappan; Yildiz, Umit Hakan; Liedberg, Bo

    2016-04-01

    Here we report on the design and synthesis of cationic water-soluble thiophene copolymers as reporters for colorimetric detection of microRNA (miRNA) in human plasma. Poly(3-alkoxythiophene) (PT) polyelectrolytes with controlled ratios of pendant groups such as triethylamine/1-methyl imidazole were synthesized for optimizing interaction with target miRNA sequence (Tseq). Incorporation of specific peptide nucleic acid (PNA) sequences with the cationic polythiophenes yielded distinguishable responses upon formation of fluorescent PT-PNA-Tseq triplex and weakly fluorescent PT-Tseq duplex, thereby enabling selective detection of target miRNA. Unlike homopolymers of PT (hPT), experimental results indicate the possibility of utilizing copolymers of PT (cPT) with appropriate ratios of pendant groups for miRNA assay in complex matrices such as plasma. As an illustration, colorimetric responses were obtained for lung cancer associated miRNA sequence (mir21) in human plasma, with a detection limit of 10 nM, illustrating the feasibility of proposed methodology for clinical applications without involving sophisticated instrumentation. The described methodology therefore possesses high potential for low-cost nucleic acid assays in resource-limited settings. PMID:26956217

  15. 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.

  16. Chemically modified nucleic acid aptamers for in vitro selections: evolving evolution.

    Science.gov (United States)

    Kusser, W

    2000-03-01

    Combinatorial library selections through the systematic evolution of ligands by exponential enrichment (SELEX) technique identify so-called nucleic acid aptamers that bind with high-affinity and specificity to a wide range of selected molecules. However, the modest chemical functionality of nucleic acids poses some limits on their versatility as binders and catalysts, and, furthermore, the sensitivity of pure RNA- and DNA-based aptamers to nucleases restricts their use as therapeutic and diagnostic agents. Here we review synthetic chemistries for modifying nucleotides that have been developed to enhance the affinity of aptamers for targets and to increase their stability in biological fluids. Implementation of in vitro selections with modified nucleotides promises to be an elegant technique for the creation of ligands with novel physical and chemical properties and is anticipated to have a significant impact on biotechnology, diagnostics and drug development. The current molecular designs and applications of modified nucleotides for in vitro selections are reviewed, along with a discussion of future developments expected to further the utility of this approach in both practical and theoretical terms. PMID:10943570

  17. THE ROLES OF LIPIDS AND NUCLEIC ACIDS IN HIV-1 ASSEMBLY

    Directory of Open Access Journals (Sweden)

    AynaAlfadhli

    2014-05-01

    Full Text Available During HIV-1 assembly, precursor Gag (PrGag proteins are delivered to plasma membrane (PM assembly sites, where they are triggered to oligomerize and bud from cells as immature virus particles. The delivery and triggering processes are coordinated by the PrGag matrix (MA and nucleocapsid (NC domains. Targeting of PrGag proteins to membranes enriched in cholesterol and phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2 is mediated by the MA domain, which also has been shown to bind both RNA and DNA. Evidence suggests that the nucleic acid-binding function of MA serves to inhibit PrGag binding to inappropriate intracellular membranes, prior to delivery to the PM. At the PM, MA domains putatively trade RNA ligands for PI(4,5P2 ligands, fostering high affinity membrane binding. Triggering of oligomerization, budding and virus particle release results when NC domains on adjacent PrGag proteins bind to viral RNA, leading to capsid (CA domain oligomerization. This process leads to the assembly of immature virus shells in which hexamers of membrane-bound MA trimers appear to organize above interlinked CA hexamers. Here we review the functions of retroviral MA proteins, with an emphasis on the nucleic acid binding capability of the HIV-1 MA protein, and its effects on membrane binding.

  18. High-throughput optical sensing of nucleic acids in a nanopore array

    Science.gov (United States)

    Huang, Shuo; Romero-Ruiz, Mercedes; Castell, Oliver K.; Bayley, Hagan; Wallace, Mark I.

    2016-01-01

    Protein nanopores such as α-hemolysin and MspA can potentially be used to sequence long strands of DNA quickly and at low cost. In order to provide high-speed sequencing, large arrays of nanopores are required that allow the nanopores to individually addressed, but current nanopore sequencing methods rely on ionic current measurements and such methods are likely to prove difficult to scale up. Here, we show that, by optically encoding the ionic flux through protein nanopores, the discrimination of nucleic acid sequences and the detection of sequence-specific nucleic acid binding events can be parallelized. We make optical recordings at a density of ~104 nanopores per mm2 in a single droplet interface bilayer. Nanopore blockades can discriminate between DNAs with sub-pA equivalent resolution, and specific miRNA sequences can be identified by differences in unzipping kinetics. By creating an array of 2500 bilayers with a micro-patterned hydrogel chip, we are also able to load different samples into specific bilayers, suitable for high-throughput nanopore recording. PMID:26322943

  19. Mechanism of Nucleic Acid Chaperone Function of Retroviral Nuceleocapsid (NC) Proteins

    Science.gov (United States)

    Rouzina, Ioulia; Vo, My-Nuong; Stewart, Kristen; Musier-Forsyth, Karin; Cruceanu, Margareta; Williams, Mark

    2006-03-01

    Recent studies have highlighted two main activities of HIV-1 NC protein contributing to its function as a universal nucleic acid chaperone. Firstly, it is the ability of NC to weakly destabilize all nucleic acid,(NA), secondary structures, thus resolving the kinetic traps for NA refolding, while leaving the annealed state stable. Secondly, it is the ability of NC to aggregate NA, facilitating the nucleation step of bi-molecular annealing by increasing the local NA concentration. In this work we use single molecule DNA stretching and gel-based annealing assays to characterize these two chaperone activities of NC by using various HIV-1 NC mutants and several other retroviral NC proteins. Our results suggest that two NC functions are associated with its zinc fingers and cationic residues, respectively. NC proteins from other retroviruses have similar activities, although expressed to a different degree. Thus, NA aggregating ability improves, and NA duplex destabilizing activity decreases in the sequence: MLV NC, HIV NC, RSV NC. In contrast, HTLV NC protein works very differently from other NC proteins, and similarly to typical single stranded NA binding proteins. These features of retroviral NCs co-evolved with the structure of their genomes.

  20. 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...

  1. Nucleic Acid Targeting: Towards Personalized Therapy for Head and Neck Cancer

    Science.gov (United States)

    Parsel, Sean M; Grandis, Jennifer R; Thomas, Sufi M

    2015-01-01

    In light of a detailed characterization of genetic aberrations in cancer, nucleic acid targeting represents an attractive therapeutic approach with significant translational potential. Head and neck squamous cell carcinoma (HNSCC) is a leading cause of cancer deaths worldwide with stagnant 5-year survival rates. Advances in conventional treatment have done little to improve survival and combined chemoradiation is associated with significant adverse effects. Recent reports have characterized the genetic alterations in HNSCC and demonstrated that mutations confer resistance to conventional and molecular targeted therapies. The ability to use specific nucleic acid sequences to inhibit cancer-associated genes including non-druggable targets facilitates personalized medicine approaches with less adverse effects. Additionally, advances in drug delivery mechanisms have increased the transfection efficiency aiding in greater therapeutic responses. Given these advances, the stage has been set to translate the information garnered from genomic studies into personalized treatment strategies. Genes involved in the tumor protein 53 (TP53) and epidermal growth factor receptor (EGFR) pathways have been extensively investigated and many promising preclinical studies have shown tumor inhibition through genetic modulation. We, and others, have demonstrated that targeting oncogene expression with gene therapy approaches is feasible in patients. Other methods such as RNA interference have proven to be effective and are potential candidates for clinical studies. This review summarizes the major advances in sequence-specific gene modulation in the preclinical setting and in clinical trials in head and neck cancer patients. PMID:26592450

  2. Construction of HA-1-DC Nucleic-acid Vaccine and Induction of Specific Cytotoxic T Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    WANG Yaya; ZHANG Donghua; HU Jinmei; LIU Wenli; ZHOU hongsheng; ZHANG Lu; LIU Dan; HUANG Zhenqian; TAN Huo

    2007-01-01

    An HA-1-DC nucleic-acid vaccine was constructed to induce anti-leukemia effect after hematopoietic stem cell transplantation (HSCT). DCs were generated from HSCT donors in vitro, and its immunologic activity was assayed by using flow cytometry and mixed lymphocytes reaction.HA-1 gene was electroporated into the cultured DCs to construct a DC nucleic-acid vaccine. After transfection for 48 h, the expression of HA-1 protein could be detected by using Western blot. The DCs were cultured with syngenic lymphocytes to induce specific cytotoxic T lymphocytes (CTLs).The cytoxicity of the CTLs was detected by LDH assay. The results showed that The DCs derived from peripheral blood monocytes (PBMCs) expressed the phenotype of DCs, and were effective in stimulating proliferation of the allogenic lymphocytes. After electroporating for 48-h, HA-1 protein was detected by using Western blot. The cytotoxity of inducing CTLs was higher than the control group. It was suggested that minor histocompatibility antigen HA-1 could be considered as a target of immunotherapy against leukemia after HSCT.

  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. 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. PMID:15008415

  5. Nucleic Acid Chaperone Activity of HIV-1 NC Proteins Investigated by Single Molecule DNA Stretching

    Science.gov (United States)

    Williams, Mark C.; Gorelick, Robert J.; Musier-Forsyth, Karin; Bloomfield, Victor A.

    2002-03-01

    HIV-1 Nucleocapsid Protein (NC) is a nucleic acid chaperone protein that is responsible for facilitating numerous nucleic acid rearrangements throughout the reverse transcription cycle of HIV-1. To understand the mechanism of NC’s chaperone function, we carried out single molecule DNA stretching studies in the presence of NC and mutant forms of NC. Using an optical tweezers instrument, we stretch single DNA molecules from the double-stranded helical state to the single-stranded (coil) state. Based on the observed cooperativity of DNA force-induced melting, we find that the fraction of melted base pairs at room temperature is increased dramatically in the presence of NC. Thus, upon NC binding, increased thermal fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations in order to find the lowest energy state. While NC destabilizes the double-stranded form of DNA, a mutant form of NC that lacks the zinc finger structures does not. DNA stretching experiments carried out in the presence of NC variants containing more subtle changes in the zinc finger structures were conducted to elucidate the contribution of each individual finger to NC’s chaperone activity, and these results will be reported.

  6. Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast.

    Science.gov (United States)

    Dong, Biqin; Almassalha, Luay M; Stypula-Cyrus, Yolanda; Urban, Ben E; Chandler, John E; Nguyen, The-Quyen; Sun, Cheng; Zhang, Hao F; Backman, Vadim

    2016-08-30

    Visualizing the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in nonperturbed, structurally and dynamically complex cellular systems, will help expand our understanding of biological processes and open the next frontier for biological discovery. Traditional superresolution techniques to visualize subdiffractional macromolecular structures formed by nucleic acids require exogenous labels that may perturb cell function and change the very molecular processes they intend to study, especially at the extremely high label densities required for superresolution. However, despite tremendous interest and demonstrated need, label-free optical superresolution imaging of nucleotide topology under native nonperturbing conditions has never been possible. Here we investigate a photoswitching process of native nucleotides and present the demonstration of subdiffraction-resolution imaging of cellular structures using intrinsic contrast from unmodified DNA based on the principle of single-molecule photon localization microscopy (PLM). Using DNA-PLM, we achieved nanoscopic imaging of interphase nuclei and mitotic chromosomes, allowing a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. This study may pave a new way for label-free superresolution nanoscopic imaging of macromolecular structures with nucleotide topologies and could contribute to the development of new DNA-based contrast agents for superresolution imaging. PMID:27535934

  7. Nucleic acid metabolism in inoculated and uninoculated gree-gram (Phaseolus aureous L.) roots during development

    International Nuclear Information System (INIS)

    Metabolism of nucleic acids in inoculated and un-inoculated green-gram roots was studied during their development in order to understand the complex physiological and biochemical interactions that take place during establishment of a normal symbiosis between a strain of Rhizobium and its legume host. Nucleic acid fractionation on MAK column at different stages of root and nodule development showed considerable changes in DNA and different RNA fractions. The proportion of DNA increased with root and nodule development. AT At nodule degenerative stage, the proportion of rRNA decreased whereas DNA and sRNS increased. Pulse labelling with 32P showed heavy labelling in mRNA region at early stage, however at 8 day stage labelling ocurred in mRNA and sRNA region in inoculated roots, while in un-inoculated roots more label appeared in lRNA and sRNA region. During active nodule stage label appeared in DNA showing the presence of mRNA-DNA hybrids. Differences in nucleotide composition of rRNA from inoculated and un-inoculated roots were also observed. (author)

  8. Spatio-Temporal Variations of High and Low Nucleic Acid Content Bacteria in an Exorheic River

    Science.gov (United States)

    Ma, Lili; Ji, Yurui; Bartlam, Mark; Wang, Yingying

    2016-01-01

    Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river. PMID:27082986

  9. Cross Talk between Cancer and Mesenchymal Stem Cells through Extracellular Vesicles Carrying Nucleic Acids

    Science.gov (United States)

    Lopatina, Tatiana; Gai, Chiara; Deregibus, Maria Chiara; Kholia, Sharad; Camussi, Giovanni

    2016-01-01

    Extracellular vesicles (EVs) are considered to be a novel complex mechanism of cell communication within the tumor microenvironment. EVs may act as vehicles for transcription factors and nucleic acids inducing epigenetic changes in recipient cells. Since tumor EVs may be present in patient biological fluids, it is important to investigate their function and molecular mechanisms of action. It has been shown that tumor cells release EVs, which are capable of regulating cell apoptosis, proliferation, invasion, and epithelial–mesenchymal transition, as well as to suppress activity of immune cells, to enhance angiogenesis, and to prepare a favorable microenvironment for metastasis. On the other hand, EVs derived from stromal cells, such as mesenchymal stem cells (MSCs), may influence the phenotype of tumor cells through reciprocal cross talk greatly influenced by the transcription factors and nucleic acids they carry. In particular, non-coding RNAs (ncRNAs), including microRNAs and long ncRNAs, have recently been identified as the main candidates for the phenotypic changes induced in the recipient cells by EVs. ncRNAs, which are important regulators of mRNA and protein expression, can function either as tumor suppressors or as oncogenes, depending on their targets. Herein, we have attempted to revise actual evidence reported in the literature on the role of EVs in tumor biology with particular regard to the cross talk of ncRNAs between cancer cells and MSCs.

  10. High-throughput optical sensing of nucleic acids in a nanopore array.

    Science.gov (United States)

    Huang, Shuo; Romero-Ruiz, Mercedes; Castell, Oliver K; Bayley, Hagan; Wallace, Mark I

    2015-11-01

    Protein nanopores such as α-haemolysin and Mycobacterium smegmatis porin A (MspA) can be used to sequence long strands of DNA at low cost. To provide high-speed sequencing, large arrays of nanopores are required, but current nanopore sequencing methods rely on ionic current measurements from individually addressed pores and such methods are likely to prove difficult to scale up. Here we show that, by optically encoding the ionic flux through protein nanopores, the discrimination of nucleic acid sequences and the detection of sequence-specific nucleic acid hybridization events can be parallelized. We make optical recordings at a density of ∼10(4) nanopores per mm(2) in a single droplet interface bilayer. Nanopore blockades can discriminate between DNAs with sub-picoampere equivalent resolution, and specific miRNA sequences can be identified by differences in unzipping kinetics. By creating an array of 2,500 bilayers with a micropatterned hydrogel chip, we are also able to load different samples into specific bilayers suitable for high-throughput nanopore recording. PMID:26322943

  11. Effects on cell growth processes (mitosis, synthesis of nucleic acids and of proteins). Chapter 7

    International Nuclear Information System (INIS)

    A review is presented of reports of the interference of -SH radioprotective agents with cell division and with the processes of nucleic acid and protein synthesis which are a prerequisite for mitosis. Mitotic activity is inhibited to the same extent in mammalian tissues as in cultures of animal and plant cells and bacteria. With cultured cells, the toxicity and the antimitotic activity have been found to be at their highest level for intermediate concentrations of the compound and to decrease for higher and lower concentrations. Inhibition of the synthesis of nucleic acids by -SH radioprotective substances has been observed with cultures of cells and bacteria and in mammalian tissues. In vitro interactions with the structures of free DNA and nucleoprotein have also been studied. The extent to which such complexes between the protective agent and DNA or nucleoprotein occur in vivo is not known. A depression of protein synthesis has been observed, and participates in the more general inhibition of growth processes. Possible mechanisms of these effects are discussed. (U.K.)

  12. Selective local lysis and sampling of live cells for nucleic acid analysis using a microfluidic probe

    Science.gov (United States)

    Kashyap, Aditya; Autebert, Julien; Delamarche, Emmanuel; Kaigala, Govind V.

    2016-01-01

    Heterogeneity is inherent to biology, thus it is imperative to realize methods capable of obtaining spatially-resolved genomic and transcriptomic profiles of heterogeneous biological samples. Here, we present a new method for local lysis of live adherent cells for nucleic acid analyses. This method addresses bottlenecks in current approaches, such as dilution of analytes, one-sample-one-test, and incompatibility to adherent cells. We make use of a scanning probe technology - a microfluidic probe - and implement hierarchical hydrodynamic flow confinement (hHFC) to localize multiple biochemicals on a biological substrate in a non-contact, non-destructive manner. hHFC enables rapid recovery of nucleic acids by coupling cell lysis and lysate collection. We locally lysed ~300 cells with chemical systems adapted for DNA or RNA and obtained lysates of ~70 cells/μL for DNA analysis and ~15 cells/μL for mRNA analysis. The lysates were introduced into PCR-based workflows for genomic and transcriptomic analysis. This strategy further enabled selective local lysis of subpopulations in a co-culture of MCF7 and MDA-MB-231 cells, validated by characteristic E-cadherin gene expression in individually extracted cell types. The developed strategy can be applied to study cell-cell, cell-matrix interactions locally, with implications in understanding growth, progression and drug response of a tumor. PMID:27411740

  13. Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters

    Science.gov (United States)

    Nott, Timothy J.; Craggs, Timothy D.; Baldwin, Andrew J.

    2016-06-01

    Membraneless organelles are cellular compartments made from drops of liquid protein inside a cell. These compartments assemble via the phase separation of disordered regions of proteins in response to changes in the cellular environment and the cell cycle. Here we demonstrate that the solvent environment within the interior of these cellular bodies behaves more like an organic solvent than like water. One of the most-stable biological structures known, the DNA double helix, can be melted once inside the liquid droplet, and simultaneously structures formed from regulatory single-stranded nucleic acids are stabilized. Moreover, proteins are shown to have a wide range of absorption or exclusion from these bodies, and can act as importers for otherwise-excluded nucleic acids, which suggests the existence of a protein-mediated trafficking system. A common strategy in organic chemistry is to utilize different solvents to influence the behaviour of molecules and reactions. These results reveal that cells have also evolved this capability by exploiting the interiors of membraneless organelles.

  14. Paper-based fluorescence resonance energy transfer assay for directly detecting nucleic acids and proteins.

    Science.gov (United States)

    Li, Hua; Fang, Xueen; Cao, Hongmei; Kong, Jilie

    2016-06-15

    Paper-based fluorescence resonance energy transfer assay (FRET) is gaining great interest in detecting macro-biological molecule. It is difficult to achieve conveniently and fast detection for macro-biological molecule. Herein, a graphene oxide (GO)-based paper chip (glass fiber) integrated with fluorescence labeled single-stranded DNA (ssDNA) for fast, inexpensive and direct detection of biological macromolecules (proteins and nucleic acids) has been developed. In this paper, we employed the Cy3/FAM-labeled ssDNA as the reporter and the GO as quencher and the original glass fiber paper as data acquisition substrates. The chip which was designed and fabricated by a cutting machine is a miniature biosensor that monitors fluorescence recovery from resonance energy transfer. The hybridization assays and fluorescence detection were all simplified, and the surface of the chip did not require immobilization or washing. A Nikon Eclipse was employed as excited resource and a commercial digital camera was employed for capturing digital images. This paper-based microfluidics chip has been applied in the detection of proteins and nucleic acids. The biosensing capability meets many potential requirements for disease diagnosis and biological analysis. PMID:26807518

  15. A Study on HSV—1Corneal Potential Infection by in Situ Nucleic Acid hybridization

    Institute of Scientific and Technical Information of China (English)

    ShaoweiLi; LixinXie

    1995-01-01

    Purpose:To evaluate the possibility of HSV-1 corneal latency by in situ nucleic acid hybridization in animal models.Methods:20 normal New Zealand White(NEW)rabbits were used,14of them were inoculated bilaterally with 3×10PFU/ml of McKrae strain HSV-1by in-trastromal injection,22/28eyes developed typical herpes simplex keratitis(HSK) diseases.At 60day postoperation(PI),4latent corneas were transplanted to one eye of 4noninfected NZW rabbits and removed2weeks PI,Corneas at all time intervals of infection and two weeks after PKPwere detected for presence of HSV-1antigen and nucleic acid sequences by using clonal IgGHSV-1antibody and biotinylated HSV-1DNAprobe individually.Results:The results showed that the HSV-1DNA sequences were retained with-in the corneal epithelium and anterior stromal keratocytes during acute diseases,while the corneas during latent infection and postoperation,the HSV-1DNAse-quences were retained only within the stromal layer with negative HSV-1antigne staining.Conclusions:These results strongly suggest that the cornea may be capable of harburing latent HSV-1.Eye Science 1995;11:117-119.

  16. DNA tetrahedron and star trigon nanostructures for target recycling detection of nucleic acid.

    Science.gov (United States)

    Li, Yueran; Chen, Xifeng; Wang, Bidou; Liu, Guangxing; Tang, Yuguo; Miao, Peng

    2016-06-01

    Human immunodeficiency virus (HIV) is a retrovirus which attacks the human body's immune system and further leads to acquired immunodeficiency syndrome (AIDS). Nucleic acid detection is of great importance in the medical diagnosis of such diseases. Herein, we develop a simple and enzyme-free electrochemical method for the target recycling detection of nuclei acid. DNA tetrahedron and star trigon nanostructures are designed and constructed on the electrode interface for target capture and signal enrichment. This strategy is convenient and sensitive, with a limit of detection as low as 1 fM, and can also successfully distinguish single-base mismatched DNA. Therefore, the proposed method has a promising potential application for HIV DNA detection. PMID:27170090

  17. Self-assembled monolayer based electrochemical nucleic acid sensor for Vibrio cholerae detection

    International Nuclear Information System (INIS)

    Nucleic acid sensor has been fabricated by immobilization of thiolated (5' end) single stranded deoxyribonucleic acid probe (ssDNA-SH) onto gold (Au) coated glass electrode for Vibriocholerae detection. This ssDNA-SH/Au bioelectrode characterized using atomic force microscopy (AFM),Fourier transforms infrared spectroscopy (FT-IR) and electrochemical technique, has been used for hybridization detection of genomic DNA (dsDNA/Au). This ssDNA-SH/Au bioelectrode can specifically detect up to 100- 500 ng/μL genomic DNA of Vibriocholeare within 60 s of hybridization time at 25°C by cyclic voltammetry (CV) using methylene blue (MB) as electro-active DNA hybridization indicator. The value of sensitivity of the dsDNA/Au electrode has been determined as 0.027μA/ng cm−2 with regression coefficient as 0.978. This DNA bioelectrode is stable for about 4 months when stored at 4°C.

  18. Liquid biopsies for liquid tumors:emerging potential of circulating free nucleic acid evaluation for the management of hematologic malignancies

    Institute of Scientific and Technical Information of China (English)

    Jay Hocking; Sridurga Mithraprabhu; Anna Kalff; Andrew Spencer

    2016-01-01

    Circulating free nucleic acids; cell free DNA and circulating micro-RNA, are found in the plasma of patients with hematologic and solid malignancies at levels higher than that of healthy individuals. In patients with hematologic malignancy cell free DNA reflects the underlying tumor mutational profile, whilst micro-RNAs reflect genetic interference mechanisms within a tumor and potentially the surrounding microenvironment and immune effector cells. These circulating nucleic acids offer a potentially simple, non-invasive, repeatable analysis that can aid in diagnosis, prognosis and therapeutic decisions in cancer treatment.

  19. The nucleic acids as early indicators of the recovery of patients subjected to total body irradiation for bone marrow transplant

    International Nuclear Information System (INIS)

    The possibility to use the concentration of nucleic acids as an early indicator for the recovery of individuals exposed to high radiation was valued in 30 patients subjected to a dose of 10 Gy (cobalt 60) in two or three sessions of total body irradiation for bone marrow transplants. The determination of the concentration of the nucleic acids was carried out prior to the irradiation, and later in different periods until the patients discharge. The behaviour of indicate such as alpha amylase serics transaminases, glicemics, alkaline phosphatase and others was also studied

  20. Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease.

    Science.gov (United States)

    Lian, Dong-Sheng; Zhao, Shu-Jin

    2016-04-01

    Rapid transmission, high morbidity, and mortality are the features of human infectious diseases caused by microorganisms, such as bacteria, fungi, and viruses. These diseases may lead within a short period of time to great personal and property losses, especially in regions where sanitation is poor. Thus, rapid diagnoses are vital for the prevention and therapeutic intervention of human infectious diseases. Several conventional methods are often used to diagnose infectious diseases, e.g. methods based on cultures or morphology, or biochemical tests based on metabonomics. Although traditional methods are considered gold standards and are used most frequently, they are laborious, time consuming, and tedious and cannot meet the demand for rapid diagnoses. Disease diagnosis using capillary electrophoresis methods has the advantages of high efficiency, high throughput, and high speed, and coupled with the different nucleic acid detection strategies overcomes the drawbacks of traditional identification methods, precluding many types of false positive and negative results. Therefore, this review focuses on the application of capillary electrophoresis based on nucleic detection to the diagnosis of human infectious diseases, and offers an introduction to the limitations, advantages, and future developments of this approach. PMID:26352354

  1. 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.

  2. Exploring the Hybridization Thermodynamics of Spherical Nucleic Acids to Tailor Probes for Diagnostic and Therapeutic Applications

    Science.gov (United States)

    Randeria, Pratik Shailesh

    Spherical nucleic acids (SNAs), three-dimensional nanoparticle conjugates composed of densely packed and highly oriented oligonucleotides around organic or inorganic nanoparticles, are an emergent class of nanostructures that show promise as single-entity agents for intracellular messenger RNA (mRNA) detection and gene regulation. SNAs exhibit superior biocompatibility and biological properties compared to linear oligonucleotides, enabling them to overcome many of the limitations of linear oligonucleotides for use in biomedical applications. However, the origins of these biologically attractive properties are not well understood. In this dissertation, the chemistry underlying one such property is studied in detail, and the findings are applied towards the rational design of more effective SNAs for diagnostic and therapeutic applications. Chapter 1 introduces the synthesis of SNAs, the unique properties that make them superior to linear nucleic acids for biomedicine, and previously studied applications of these structures. Chapter 2 focuses on quantitatively studying the impact of the chemical structure of the SNA on its ability to hybridize multiple complementary nucleic acids. This chapter lays the groundwork for understanding the factors that govern SNA hybridization thermodynamics and how to tailor SNAs to increase their binding affinity to target mRNA strands. Chapters 3 and 4 capitalize on this knowledge to engineer probes for intracellular mRNA detection and gene regulation applications. Chapter 3 reports the development of an SNA-based probe that can simultaneously report the expression level of two different mRNA transcripts in live cells and differentiate diseased cells from non-diseased cells. Chapter 4 investigates the use of topically-applied SNAs to down-regulate a critical mediator of impaired wound healing in diabetic mice to accelerate wound closure. This study represents the first topical therapeutic application of SNA nanotechnology to treat open

  3. Nucleic acid analysis using liquid chromatography and electrospray ionization mass spectrometry

    International Nuclear Information System (INIS)

    The development of fast, reliable and sensitive methods for the analysis of biomolecules, especially nucleic acids, proteins, and peptides, is at the same time a deciding motor and has to keep pace with the steady progress of biochemical, molecular biological, and medical science. Miniaturized chromatographic separation methods are frequently the method of choice for the separation and characterization of complex biopolymer mixtures, when the amount of sample is limited. The concept of monolithic chromatographic separation media is especially favorable for the fabrication of capillary columns. Chemical immobilization of the monolith at the capillary wall eliminates the tedious preparation of retaining frits; the continuous porous polymer exhibits favorable mass transfer properties, enabling excellent separation efficiency. We prepared monolithic columns within the confines of fused silica capillaries of 200 μm i.d. by copolymerization of styrene and divinylbenzene in the presence of a suitable porogen mixture of 1-decanol and tetrahydrofuran. Different chemical and physical parameters that influence the morphology, porosity and separation efficiency of the obtained monolithic chromatographic columns were investigated, specifically the influence of the amount of cross-linking monomer, of different porogens and porogen mixtures, of polymerization temperature, and finally of the amount of radical initiator. The prepared monolithic columns were characterized by measurement of permeability with different solvents, determination of the pore size distribution by inverse size exclusion chromatography, examination of the morphology by scanning electron micrographs, and finally by chromatographic testing and determination of the separation parameters. Finally, the developed separation systems were applied to the separation of DNA ladder markers, of PCR amplified DNA fragments containing sequence-tagged sites used in the genotyping of individuals and of amplified short

  4. Culture confirmation of gonococcal infection by recall of subjects found to be positive by nucleic acid amplification tests in general practice

    DEFF Research Database (Denmark)

    Møller, Jens Kjølseth

    2010-01-01

    To evaluate a routine notification of general practitioners to recall nucleic acid amplification test (NAAT)-positive subjects for culture of Neisseria gonorrhoeae to confirm gonococcal infection in the community.......To evaluate a routine notification of general practitioners to recall nucleic acid amplification test (NAAT)-positive subjects for culture of Neisseria gonorrhoeae to confirm gonococcal infection in the community....

  5. DMPD: The role of viral nucleic acid recognition in dendritic cells for innate andadaptive antiviral immunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18086372 The role of viral nucleic acid recognition in dendritic cells for innate a...1-14. Epub 2007 Nov 9. (.png) (.svg) (.html) (.csml) Show The role of viral nucleic acid recognition in dend...ritic cells for innate andadaptive antiviral immunity. PubmedID 18086372 Title The

  6. Nucleic Acids in Human Glioma Treatment: Innovative Approaches and Recent Results

    Directory of Open Access Journals (Sweden)

    S. Catuogno

    2012-01-01

    Full Text Available Gliomas are the most common primary central nervous system tumors with a dismal prognosis. Despite recent advances in surgery, radiotherapy, and chemotherapy, current treatment regimens have a modest survival benefit. A crucial challenge is to deliver drugs effectively to invasive glioma cells residing in a sanctuary within the central nervous system. New therapies are essential, and oligonucleotide-based approaches, including antisense, microRNAs, small interfering RNAs, and nucleic acid aptamers, may provide a viable strategy. Thanks to their unique characteristics (low size, good affinity for the target, no immunogenicity, chemical structures that can be easily modified to improve their in vivo applications, these molecules may represent a valid alternative to antibodies particularly to overcome challenges presented by the blood-brain barrier. Here we will discuss recent results on the use of oligonucleotides that will hopefully provide new effective treatment for gliomas.

  7. Characterization of the interaction between collectin 11 (CL-11, CL-K1) and nucleic acids

    DEFF Research Database (Denmark)

    Henriksen, Maiken Lumby; Brandt, Jette; Iyer, Sinduja S C;

    2013-01-01

    Collectins are a group of innate immune proteins that contain collagen-like regions and globular C-type lectin domains. Via the lectin domains, collectins recognize and bind to various microbial carbohydrate patterns. Collectin 11 (CL-11) exists in complex with the complement activating MBL......-associated proteases, MASPs. In the present work, we characterize the interaction between CL-11 and DNA, and show that CL-11 binds to DNA from a variety of origins in a calcium-independent manner. CL-11 binds also to apoptotic cells presenting extracellular DNA on their surface. The binding to DNA is sensitive to...... changes in ionic strength and pH. Competition studies show that CL-11 binds to nucleic acids and carbohydrates via separate binding-sites and oligomericity appears crucial for binding activity. Combined interaction with DNA and mannan strongly increases binding avidity. By surface plasmon resonance we...

  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....... Consistent with the extract experiments, treatment with adduct forming PNA conjugates (psoralen and chlorambucil) resulted in a decrease in background correction frequencies in transiently transfected cells, whereas unmodified PNA or the PNA-camptothecin conjugate had little or no effect. These results...... suggest that simple triplex forming PNAs have little effect on proximal gene correctional events whereas PNA conjugates capable of forming DNA adducts and interstrand crosslinks are strong inhibitors. Most interestingly the PNA conjugated to the topoisomerase inhibitor, camptothecin enhanced repair in...

  9. Simulated Raman correlation spectroscopy for quantifying nucleic acid-silver composites

    Science.gov (United States)

    Freeman, Lindsay M.; Smolyaninov, Alexei; Pang, Lin; Fainman, Yeshaiahu

    2016-03-01

    Plasmonic devices are of great interest due to their ability to confine light to the nanoscale level and dramatically increase the intensity of the electromagnetic field, functioning as high performance platforms for Raman signal enhancement. While Raman spectroscopy has been proposed as a tool to identify the preferential binding sites and adsorption configurations of molecules to nanoparticles, the results have been limited by the assumption that a single binding site is responsible for molecular adsorption. Here, we develop the simulated Raman correlation spectroscopy (SRCS) process to determine which binding sites of a molecule preferentially bind to a plasmonic material and in what capacity. We apply the method to the case of nucleic acids binding to silver, discovering that multiple atoms are responsible for adsorption kinetics. This method can be applied to future systems, such as to study the molecular orientation of adsorbates to films or protein conformation upon adsorption.

  10. Nucleic acid and protein synthesis during lateral root initiation in Marsilea quadrifolia (Marsileaceae)

    Science.gov (United States)

    Lin, B. L.; Raghavan, V.

    1991-01-01

    The pattern of DNA, RNA, and protein synthesis during lateral root initiation in Marsilea quadrifolia L. was monitored by autoradiography of incorporated of 3H-thymidine, 3H-uridine, and 3H-leucine, respectively. DNA synthesis was associated with the enlargement of the lateral root initial prior to its division. Consistent with histological studies, derivatives of the lateral root initial as well as the cells of the adjacent inner cortex and pericycle of the parent root also continued to synthesize DNA. RNA and protein synthetic activities were found to be higher in the lateral root initials than in the endodermal initials of the same longitudinal layer. The data suggest a role for nucleic acid and protein synthesis during cytodifferentiation of a potential endodermal cell into a lateral root initial.

  11. Inhibition of miR-21 in glioma cells using catalytic nucleic acids.

    Science.gov (United States)

    Belter, Agnieszka; Rolle, Katarzyna; Piwecka, Monika; Fedoruk-Wyszomirska, Agnieszka; Naskręt-Barciszewska, Mirosława Z; Barciszewski, Jan

    2016-01-01

    Despite tremendous efforts worldwide, glioblastoma multiforme (GBM) remains a deadly disease for which no cure is available and prognosis is very bad. Recently, miR-21 has emerged as a key omnipotent player in carcinogenesis, including brain tumors. It is recognized as an indicator of glioma prognosis and a prosperous target for anti-tumor therapy. Here we show that rationally designed hammerhead ribozymes and DNAzymes can target miR-21 and/or its precursors. They decrease miR-21 level, and thus silence this oncomiR functions. We demonstrated that anti-miRNA catalytic nucleic acids show a novel terrific arsenal for specific and effective combat against diseases with elevated cellular miR-21 content, such as brain tumors. PMID:27079911

  12. ESR spin elimination research on reaction of triplet state of VK3 with nucleic acid derivatives

    International Nuclear Information System (INIS)

    In order to study the reaction of triplet state of VK3 with nucleic acid derivatives, the ESR (Electron spin resonance) spin elimination has been used with 4-oxo-TEMPO as spin trap. The reactivities of 3VK3* with nucleosides, nucleotides, and models of telomeric DNA, template of telomerase RNA and its L6-P6 region have been confirmed respectively. The results show that the tendency of reaction of 3VK3* with nucleosides is Gua>Ade>Cyt>Thy, meanwhile dGMP>dAMP>dCMP> TMP for reaction of 3VK3* with deoxy-nucleotides, and the one for reaction of 3VK3* with telomeric DNA is the highest in polynucleotides. This is highly accordant with both of their corresponding rate constants determined by laser flash photolysis and their oxidation-reduction potentials. It also shows that the reactivity of 3VK3* with polynucleotides is directly proportional to their G amount. (authors)

  13. Tailored host-guest lipidic cubic phases: a protocell model exhibiting nucleic acid recognition.

    Science.gov (United States)

    Komisarski, Marek; Osornio, Yazmin M; Siegel, Jay S; Landau, Ehud M

    2013-01-21

    A classical conundrum in origin-of-life studies relates to the nature of the first chemical system: was it a carrier of genetic information or a facilitator of cellular compartmentalization? Here we present a system composed of tailor-made nucleolipids and hydrated monoolein, which assemble at ambient temperatures to form host-guest lipidic cubic phase (LCP) materials that are stable in bulk water and can perform both functions. As such, they may represent a molecular model for a protocell in origin-of-life studies. Nucleolipids within the lipidic material sequester and bind selectively complementary oligonucleotide sequences from solution by virtue of base-pairing; noncomplementary sequences diffuse freely between the LCP material and the bulk aqueous environment. Sequence specific enrichment of nucleic acids within the LCP material demonstrates an effective mechanism for selection of genetic material in these cell-mimetic systems. PMID:23239006

  14. Electrostatic Binding and Hydrophobic Collapse of Peptide-Nucleic Acid Aggregates Quantified Using Force Spectroscopy

    CERN Document Server

    Camunas-Soler, Joan; Bizarro, Cristiano V; de Loreno, Sara; Fuentes-Perez, Maria Eugenia; Ramsch, Roland; Vilchez, Susana; Solans, Conxita; Moreno-Herrero, Fernando; Albericio, Fernando; Eritja, Ramon; Giralt, Ernest; Dev, Sukhendu B; Ritort, Felix

    2014-01-01

    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 which 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 interact...

  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. Fractionation of SWNT/nucleic acid complexes by agarose gel electrophoresis

    International Nuclear Information System (INIS)

    We show that aqueous dispersions of single-walled carbon nanotubes (SWNTs), prepared with the aid of nucleic acids (NAs) such as RNA or DNA, can be separated into fractions using agarose gel electrophoresis. In a DC electric field, SWNT/NA complexes migrate in the gel in the direction of positive potential to form well-defined bands. Raman spectroscopy as a function of band position shows that nanotubes having different spectroscopic properties possess different electrophoretic mobilities. The migration patterns for SWNT/RNA and SWNT/DNA complexes differ. Parallel elution of the SWNT/NA complexes from the gel during electrophoresis and subsequent characterization by AFM reveals differences in nanotube diameter, length and curvature. The results suggest that fractionation of nanotubes can be achieved by this procedure. We discuss factors affecting the mobility of the nanotube complexes and propose analytical applications of this technique

  17. Enhanced cellular delivery of cell-penetrating peptide-peptide nucleic acid conjugates by photochemical internalization

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2011-01-01

    Cell-penetrating peptides (CPPs) have been widely used for a cellular delivery of biologically relevant cargoes including antisense peptide nucleic acids (PNAs). Although chemical conjugation of PNA to a variety of CPPs significantly improves the cellular uptake of the PNAs, bioavailability...... (antisense activity) is still limited by endocytotic entrapment. We have shown that this low bioavailability can be greatly improved by combining CPP-PNA conjugate administration with a photochemical internalization technique using photosensitizers such as aluminum phthalocyanine (AlPcS(2a)) or...... cellular efficacy of CPP conjugates were evaluated by measuring luciferase activity as a result of splicing correction and was also confirmed by RT-PCR analysis of luciferase pre-mRNA....

  18. Isothermal cycling and cascade signal amplification strategy for ultrasensitive colorimetric detection of nucleic acids

    International Nuclear Information System (INIS)

    We have designed a novel isothermal cascade signal-amplification strategy for ultrasensitive colorimetric determination of nucleic acids. It is based on double-cycling amplification with formation of DNAzyme via a polymerase-induced strand-displacement reaction and nicking endonuclease-assisted recycling. The assay makes use of a hairpin DNA, a short primer, KF-polymerase, and nicking endonuclease. The presence of a target DNA triggers the strand-displacement and polymerization reaction with the formation of numerous DNAzyme molecules. Upon addition of H2O2 to the resulting mixture, the H2O2 reacts with 2,2′-azino-bis (3-ethylbenzothiozoline)-6-sulfonate to form a colored product in the aid of DNAzyme, which is quantified by photometry at 415 nm. Under optimal conditions, the assay allows target DNA to be determined at concentration as low as 0.6 aM. (author)

  19. Redox labelling of nucleic acids for analyzing nucleotide sequences and monitoring DNA-protein interactions

    Czech Academy of Sciences Publication Activity Database

    Fojta, Miroslav; Havran, Luděk; Horáková Brázdilová, Petra; Pivoňková, Hana; Kostečka, Pavel; Macíčková-Cahová, Hana; Raindlová, Veronika; Vrábel, Milan; Hocek, Michal

    Praha : Institute of Organic Chemistry and Biochemistry AS CR, v. v. i., 2011 - (Hocek, M.), s. 155-158 ISBN 978-80-86241-37-1. - (Collection Symposium Series. 12). [Chemistry of Nucleic Acid Components /15./. Český Krumlov (CZ), 05.06.2011-10.06.2011] R&D Projects: GA MŠk LC512; GA MŠk(CZ) LC06035; GA AV ČR(CZ) IAA400040901; GA AV ČR(CZ) IAA400040903; GA ČR GA203/09/0317; GA AV ČR(CZ) GPP206/11/P739 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA * electrochemistry * redox labeling Subject RIV: CC - Organic Chemistry

  20. Coiled coil interactions for the targeting of liposomes for nucleic acid delivery

    Science.gov (United States)

    Oude Blenke, Erik E.; van den Dikkenberg, Joep; van Kolck, Bartjan; Kros, Alexander; Mastrobattista, Enrico

    2016-04-01

    Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner.Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes

  1. Sequence-selective targeting of duplex DNA by peptide nucleic acids

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2010-01-01

    nucleic acid (PNA) can recognize duplex DNA with high sequence specificity and affinity in triplex, duplex and double-duplex invasive modes or non-invasive triplex modes. Novel PNA modification has improved the affinity for DNA recognition via duplex invasion, double-duplex invasion and triplex......Sequence-selective gene targeting constitutes an attractive drug-discovery approach for genetic therapy, with the aim of reducing or enhancing the activity of specific genes at the transcriptional level, or as part of a methodology for targeted gene repair. The pseudopeptide DNA mimic peptide...... recognition considerably. Such modifications have also resulted in new approaches to targeted gene repair and sequence-selective double-strand cleavage of genomic DNA....

  2. Interaction of anticancer drug methotrexate with nucleic acids analyzed by multi-spectroscopic method

    Science.gov (United States)

    Cai, Changqun; Chen, Xiaoming; Gong, Hang

    2009-02-01

    Methotrexate (MTX) as an antifolate, which is widely used as chemotherapeutic drugs. A high-dose MTX therapy has a direct toxicity influence on the non-germinal cells, especially the liver cells. It is known that the inject dose for adults is 10-30 mg and is half for children for routine use, while our experiments showed that the optimum dosage of MTX which enhanced the RLS intensities to the maximum is 4.54 ng ml -1. The interaction of methotrexate (MTX) with nucleic acids in aqueous solution in the presence of cetyltrimethylammonium bromide (CTMAB), a kind of cationic surfactant similar to the Human cells, were investigated based on the measurements of resonance light scattering (RLS), UV-vis, fluorescence and NMR spectra, etc. The interaction has been proved to give a ternary complex of MTX-CTMAB-DNA in BR buffer (pH 9.30), which exhibits strong enhanced RLS signals at 339.5 nm.

  3. 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

    both PCR and real-time PCR, the addition of LNA showed blocking of the amplification of genomic XBP1 but not cDNA XBP1. To test the effect of melting temperature (Tm) on the LNA, we investigated the number of LNA nucleotides that could be replaced with DNA nucleotides and still retain the blocking......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...... activity. More than three DNA nucleotides reduced the LNA inhibition ability. The sequence specificity of the LNA was tested by investigating the number of LNA nucleotide mismatches permitted. The introduction of one mismatch maintained the inhibition of genomic amplification whereas two mismatches reduced...

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

    DEFF Research Database (Denmark)

    Vollmer, Jörg; Jepsen, Jan Stenvang; Uhlmann, Eugen; Schetter, Christian; Jurk, Marion; Wader, Tanja; Wüllner, Meike; Krieg, Arthur M; Stenvang, Jan

    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...... been shown to possess immune modulatory capacities. We investigated the effects of LNA substitutions on immune stimulation mediated by antisense ODN G3139 or CpG ODN 2006. LNA ODNs were tested for their ability to stimulate cytokine secretion from human immune cells or TLR9-dependent signaling....... Phosphorothioate chimeric LNA/DNA antisense ODNs with phosphodiester-linked LNA nucleobases at both ends showed a marked decrease of immune modulation with an increasing number of 3' and 5' LNA bases. In addition, guanosine-LNA and cytosine-LNA or simply cytosine-LNA substitutions in the CpG dinucleotides of ODN...

  5. Software scripts for quality checking of high-throughput nucleic acid sequencers.

    Science.gov (United States)

    Lazo, G R; Tong, J; Miller, R; Hsia, C; Rausch, C; Kang, Y; Anderson, O D

    2001-06-01

    We have developed a graphical interface to allow the researcher to view and assess the quality of sequencing results using a series of program scripts developed to process data generated by automated sequencers. The scripts are written in Perl programming language and are executable under the cgibin directory of a Web server environment. The scripts direct nucleic acid sequencing trace file data output from automated sequencers to be analyzed by the phred molecular biology program and are displayed as graphical hypertext mark-up language (HTML) pages. The scripts are mainly designed to handle 96-well microtiter dish samples, but the scripts are also able to read data from 384-well microtiter dishes 96 samples at a time. The scripts may be customized for different laboratory environments and computer configurations. Web links to the sources and discussion page are provided. PMID:11414222

  6. Simulated Raman correlation spectroscopy for quantifying nucleic acid-silver composites

    Science.gov (United States)

    Freeman, Lindsay M.; Smolyaninov, Alexei; Pang, Lin; Fainman, Yeshaiahu

    2016-01-01

    Plasmonic devices are of great interest due to their ability to confine light to the nanoscale level and dramatically increase the intensity of the electromagnetic field, functioning as high performance platforms for Raman signal enhancement. While Raman spectroscopy has been proposed as a tool to identify the preferential binding sites and adsorption configurations of molecules to nanoparticles, the results have been limited by the assumption that a single binding site is responsible for molecular adsorption. Here, we develop the simulated Raman correlation spectroscopy (SRCS) process to determine which binding sites of a molecule preferentially bind to a plasmonic material and in what capacity. We apply the method to the case of nucleic acids binding to silver, discovering that multiple atoms are responsible for adsorption kinetics. This method can be applied to future systems, such as to study the molecular orientation of adsorbates to films or protein conformation upon adsorption. PMID:27010074

  7. Direct observation of transition paths during the folding of proteins and nucleic acids.

    Science.gov (United States)

    Neupane, Krishna; Foster, Daniel A N; Dee, Derek R; Yu, Hao; Wang, Feng; Woodside, Michael T

    2016-04-01

    Transition paths, the fleeting trajectories through the transition states that dominate the dynamics of biomolecular folding reactions, encapsulate the critical information about how structure forms. Owing to their brief duration, however, they have not previously been observed directly. We measured transition paths for both nucleic acid and protein folding, using optical tweezers to observe the microscopic diffusive motion of single molecules traversing energy barriers. The average transit times and the shapes of the transit-time distributions agreed well with theoretical expectations for motion over the one-dimensional energy landscapes reconstructed for the same molecules, validating the physical theory of folding reactions. These measurements provide a first look at the critical microscopic events that occur during folding, opening exciting new avenues for investigating folding phenomena. PMID:27124461

  8. Comparative Molecular Mechanics and Quantum Mechanics Study of Microhydration of Nucleic Acid Bases

    CERN Document Server

    Lino, J; Deriabina, A; Velasco, M; Poltev, V

    2013-01-01

    DNA is the most important biological molecule, and its hydration contributes essentially to the structure and functions of the double helix. We analyze the microhydration of the individual bases of nucleic acids and their methyl derivatives using methods of molecular mechanics (MM) with the Poltev-Malenkov (PM), AMBER and OPLS force fields, as well as ab initio Quantum Mechanics (QM) calculations at MP2/6-31G(d,p) level of theory. A comparison is made between the calculated interaction energies and the experimental enthalpies of microhydration of bases, obtained from mass spectrometry at low temperatures. Each local water-base interaction energy minimum obtained with MM corresponds to the minimum obtained with QM. General qualitative agreement was observed in the geometrical characteristics of the local minima obtained via the two groups of methods. MM minima correspond to slightly more coplanar structures than those obtained via QM methods, and the absolute MM energy values overestimate corresponding values ...

  9. Accrued somatic mutations (nucleic acid changes) trigger ALS: 2005-2015 update.

    Science.gov (United States)

    Armon, Carmel

    2016-06-01

    Amyotrophic lateral sclerosis (ALS) is a multilevel disease of the motor neuron system. The mechanisms triggering disease onset should be considered separately from those facilitating its spread and motor neuron death. In 2005, I brought together clinical and epidemiological evidence to support the hypothesis that acquired nucleic acid changes may trigger sporadic ALS. Since 2005, the conceptual foundations for this hypothesis have been strengthened. The journal Amyotrophic Lateral Sclerosis was renamed Amyotrophic Lateral Sclerosis & Frontotemporal Degeneration. The focal onset, with simultaneous initial maximal upper and lower motor neuron involvement in the region of onset, and patterns of spread, were characterized further. Clues from the epidemiology of sporadic ALS were affirmed by quantitative analysis, including the increase in disease incidence with age, suggesting accrual of time-dependent changes, and the confirmation of smoking as an established risk factor. Additional observations support the conclusion that accrued somatic mutations trigger onset of ALS. Muscle Nerve 53: 842-849, 2016. PMID:26799358

  10. Aptamer- and nucleic acid enzyme-based systems for simultaneous detection of multiple analytes

    Science.gov (United States)

    Lu, Yi; Liu, Juewen

    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.

  11. 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...... targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild...... 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...

  12. Fractionation of SWNT/nucleic acid complexes by agarose gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Vetcher, Alexandre A [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States); Srinivasan, Srimeenakshi [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States); Vetcher, Ivan A [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States); Abramov, Semen M [NanoTech Institute, University of Texas at Dallas, Richardson, TX 75083 (United States); Kozlov, Mikhail [NanoTech Institute, University of Texas at Dallas, Richardson, TX 75083 (United States); Baughman, Ray H [NanoTech Institute, University of Texas at Dallas, Richardson, TX 75083 (United States); Levene, Stephen D [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States)

    2006-08-28

    We show that aqueous dispersions of single-walled carbon nanotubes (SWNTs), prepared with the aid of nucleic acids (NAs) such as RNA or DNA, can be separated into fractions using agarose gel electrophoresis. In a DC electric field, SWNT/NA complexes migrate in the gel in the direction of positive potential to form well-defined bands. Raman spectroscopy as a function of band position shows that nanotubes having different spectroscopic properties possess different electrophoretic mobilities. The migration patterns for SWNT/RNA and SWNT/DNA complexes differ. Parallel elution of the SWNT/NA complexes from the gel during electrophoresis and subsequent characterization by AFM reveals differences in nanotube diameter, length and curvature. The results suggest that fractionation of nanotubes can be achieved by this procedure. We discuss factors affecting the mobility of the nanotube complexes and propose analytical applications of this technique.

  13. Coiled coil interactions for the targeting of liposomes for nucleic acid delivery

    Science.gov (United States)

    Oude Blenke, Erik E.; van den Dikkenberg, Joep; van Kolck, Bartjan; Kros, Alexander; Mastrobattista, Enrico

    2016-04-01

    Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner.Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes

  14. 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

  15. An air-pressure-free elastomeric valve for integrated nucleic acid analysis by capillary electrophoresis

    Science.gov (United States)

    Jung, Wooseok; Barrett, Matthew; Brooks, Carla; Rivera, Andrew; Birdsell, Dawn N.; Wagner, David M.; Zenhausern, Frederic

    2015-12-01

    We present a new elastomeric valve for integrated nucleic acid analysis by capillary electrophoresis. The valve functions include metering to capture a designated volume of biological sample into a polymerase chain reaction (PCR) chamber, sealing to preserve the sample during PCR cycling, and transfer of the PCR-products and on-chip formamide post-processing for the analysis of DNA fragments by capillary gel electrophoresis. This new valve differs from prior art polydimethylsiloxane (PDMS) valves in that the valve is not actuated externally by air-pressure or vacuum so that it simplifies a DNA analysis system by eliminating the need for an air-pressure or vacuum source, and off-cartridge solenoid valves, control circuit boards and software. Instead, the new valve is actuated by a thermal cycling peltier assembly integrated within the hardware instrument that tightly comes in contact with a microfluidic cartridge for thermal activation during PCR, so that it spontaneously closes the valve without an additional actuator system. The valve has bumps in the designated locations so that it has a self-alignment that does not require precise alignment of a valve actuator. Moreover, the thickness of the new valve is around 600 μm with an additional bump height of 400 μm so that it is easy to handle and very feasible to fabricate by injection molding compared to other PDMS valves whose thicknesses are around 30-100 μm. The new valve provided over 95% of metering performance in filling the fixed volume of the PCR chamber, preserved over 97% of the sample volume during PCR, and showed very comparable capillary electrophoresis peak heights to the benchtop assay tube controls with very consistent transfer volume of the PCR-product and on-chip formamide. The new valve can perform a core function for integrated nucleic acid analysis by capillary electrophoresis.

  16. An air-pressure-free elastomeric valve for integrated nucleic acid analysis by capillary electrophoresis

    International Nuclear Information System (INIS)

    We present a new elastomeric valve for integrated nucleic acid analysis by capillary electrophoresis. The valve functions include metering to capture a designated volume of biological sample into a polymerase chain reaction (PCR) chamber, sealing to preserve the sample during PCR cycling, and transfer of the PCR-products and on-chip formamide post-processing for the analysis of DNA fragments by capillary gel electrophoresis. This new valve differs from prior art polydimethylsiloxane (PDMS) valves in that the valve is not actuated externally by air-pressure or vacuum so that it simplifies a DNA analysis system by eliminating the need for an air-pressure or vacuum source, and off-cartridge solenoid valves, control circuit boards and software. Instead, the new valve is actuated by a thermal cycling peltier assembly integrated within the hardware instrument that tightly comes in contact with a microfluidic cartridge for thermal activation during PCR, so that it spontaneously closes the valve without an additional actuator system. The valve has bumps in the designated locations so that it has a self-alignment that does not require precise alignment of a valve actuator. Moreover, the thickness of the new valve is around 600 μm with an additional bump height of 400 μm so that it is easy to handle and very feasible to fabricate by injection molding compared to other PDMS valves whose thicknesses are around 30–100 μm. The new valve provided over 95% of metering performance in filling the fixed volume of the PCR chamber, preserved over 97% of the sample volume during PCR, and showed very comparable capillary electrophoresis peak heights to the benchtop assay tube controls with very consistent transfer volume of the PCR-product and on-chip formamide. The new valve can perform a core function for integrated nucleic acid analysis by capillary electrophoresis. (paper)

  17. Urinary markers of nucleic acid oxidation in Danish overweight/obese children and youths.

    Science.gov (United States)

    Kloppenborg, Julie Tonsgaard; Fonvig, Cilius Esmann; Johannesen, Jesper; Bjerrum, Poul Jannik; Poulsen, Henrik Enghusen; Holm, Jens-Christian

    2016-07-01

    Urinary excretion of the RNA and DNA oxidation markers, 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in newly diagnosed adult type 2 diabetics are reported to be long-term predictors of mortality independent of conventional risk factors. In the current 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. Forty-two (24 girls) overweight and 35 lean (19 girls) children and adolescents were recruited from the Registry of the Danish Childhood Obesity Biobank. Anthropometric measurements were collected at baseline and glucose metabolism was assessed by an oral glucose tolerance test. A urine sample was obtained during the test. Linear regression did not demonstrate any associations between the urinary markers and the degree of obesity 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 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 tolerance seem to exhibiting an increased oxidative stress level, but due to the small sample size in this study, further investigations are required to elucidate this correlation. PMID:26982114

  18. Sensitive electrochemical monitoring of nucleic acids coupling DNA nanostructures with hybridization chain reaction

    Energy Technology Data Exchange (ETDEWEB)

    Zhuang, Junyang; Fu, Libing; Xu, Mingdi; Yang, Huanghao; Chen, Guonan; Tang, Dianping, E-mail: dianping.tang@fzu.edu.cn

    2013-06-14

    Graphical abstract: -- Highlights: •A new signal-on metallobioassay was developed for detection of nucleic acids. •Target-triggered long-range self-assembled DNA nanostructures are used for amplification of electronic signal. •Hybridization chain reaction is utilized for construction of long-range DNA nanostructures. -- Abstract: Methods based on metal nanotags have been developed for metallobioassay of nucleic acids, but most involve complicated labeling or stripping procedures and are unsuitable for routine use. Herein, we report the proof-of-concept of a novel and label-free metallobioassay for ultrasensitive electronic determination of human immunodeficiency virus (HIV)-related gene fragments at an ultralow concentration based on target-triggered long-range self-assembled DNA nanostructures and DNA-based hybridization chain reaction (HCR). The signal is amplified by silver nanotags on the DNA duplex. The assay mainly consists of capture probe, detection probe, and two different DNA hairpins. In the presence of target DNA, the capture probe immobilized on the sensor sandwiches target DNA with the 3′ end of detection probe. Another exposed part of detection probe at the 5′ end opens two alternating DNA hairpins in turn, and propagates a chain reaction of hybridization events to form a nicked double-helix. Finally, numerous silver nanotags are immobilized onto the long-range DNA nanostructures, each of which produces a strong electronic signal within the applied potentials. Under optimal conditions, the target-triggered long-range DNA nanostructures present good electrochemical behaviors for the detection of HIV DNA at a concentration as low as 0.5 fM. Importantly, the outstanding sensitivity can make this approach a promising scheme for development of next-generation DNA sensors without the need of enzyme labeling or fluorophore labeling.

  19. Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Qualley, Dominic F., E-mail: dqualley@berry.edu; Sokolove, Victoria L.; Ross, James L.

    2015-03-13

    Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two other retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable. - Highlights: • BLV NC binds strongly to DNA and RNA. • BLV NC promotes mini-TAR annealing as well as HIV-1 NC. • Annealing kinetics suggest a low degree of similarity between BLV NC and HTLV-1 NC.

  20. Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

    International Nuclear Information System (INIS)

    Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two other retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable. - Highlights: • BLV NC binds strongly to DNA and RNA. • BLV NC promotes mini-TAR annealing as well as HIV-1 NC. • Annealing kinetics suggest a low degree of similarity between BLV NC and HTLV-1 NC

  1. Genetic studies in varicocele infertility: I: pedigree, nuclear sex and seminal nucleic acids.

    Science.gov (United States)

    Mahmoud, K Z; Hafeiz, A R; Salam, E A; El Kerdasi, Z; Aiad, F

    1983-01-01

    The hereditary predisposition of varicocele men to develop infertility was investigated in a controlled study involving 30 infertile varicocele patients. Analysis of the pedigree charts, determination of sex chromatin constitution, and biochemical measurement of seminal nucleic acid (DNA/RNA) were undertaken. Sibs and Kins of infertile varicocele patients showed various developmental anomalies particularly of the genito-crural area. cocele formation while the gonads develop in the normal line. Such condition may be represented in fertile men with clinically detectable varicocele. In addition, the postulated defective gene in VI patients may interfere as well with the normal development of the mesenchyme-derived interstitial cells of Leydig. Defective testicular steroid hormone biosynthesis thus obtained may explain the reported decrease in androgen level in peripheral blood, sexual impotentia and accessory genital gland dysfunction (Raboch and Starka - 1971; Comhaire and Vermeulen - 1975). Moreover, being androgen dependent (Lacy - 1973), spermatogenesis may display abnormal patterns due to lowered intratesticular androgen concentration (Rodriguez-Rigau et al. - 1978). The outcome and severity of spermatogenic derangement may depend partially on the severity of disordered androgen biosynthesis. The present work showed significant decrease in sperm count, motility, normal forms and nucleic acid content (DNA and RNA) of the seminal fluid in VI patients. The decrease in the amount of DNA per millilitre of seminal fluid in VI patients may be explained by the decrease in sperm population (oligozoospermia). The decrease in the amount of RNA per millilitre of seminal fluid in VI patients may be due to the total decrease in cellular content of the seminal fluid.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6198939

  2. Circulating nucleic acids in plasma and serum (CNAPS: applications in oncology

    Directory of Open Access Journals (Sweden)

    González-Masiá JA

    2013-07-01

    Full Text Available José A González-Masiá,1 Damián García-Olmo,2 Dolores C García-Olmo31General Surgery Service, General University Hospital of Albacete, Albacete, 2Department of Surgery, Universidad Autónoma de Madrid and La Paz University Hospital, IdiPaz, Madrid, 3Experimental Research Unit, General University Hospital of Albacete, Albacete, SpainAbstract: The presence of small amounts of circulating nucleic acids in plasma and serum (CNAPS is not a new finding. The verification that such amounts are significantly increased in cancer patients, and that CNAPS might carry a variety of genetic and epigenetic alterations related to cancer development and progression, has aroused great interest in the scientific community in the last decades. Such alterations potentially reflect changes that occur during carcinogenesis, and include DNA mutations, loss of heterozygosity, viral genomic integration, disruption of microRNA, hypermethylation of tumor suppressor genes, and changes in the mitochondrial DNA. These findings have led to many efforts toward the implementation of new clinical biomarkers based on CNAPS analysis. In the present article, we review the main findings related to the utility of CNAPS analysis for early diagnosis, prognosis, and monitoring of cancer, most of which appear promising. However, due to the lack of harmonization of laboratory techniques, the heterogeneity of disease progression, and the small number of recruited patients in most of those studies, there has been a poor translation of basic research into clinical practice. In addition, many aspects remain unknown, such as the release mechanisms of cell-free nucleic acids, their biological function, and the way by which they circulate in the bloodstream. It is therefore expected that in the coming years, an improved understanding of the relationship between CNAPS and the molecular biology of cancer will lead to better diagnosis, management, and treatment.Keywords: plasma, cancer, clinical

  3. Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

    Energy Technology Data Exchange (ETDEWEB)

    Koopman, R P; Langlois, R G; Nasarabadi, S; Benett, W J; Colston, B W; Johnson, D C; Brown, S B; Stratton, P L; Milanovich, F P

    2002-04-17

    This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flow cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.

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

    Science.gov (United States)

    Broedbaek, Kasper; Siersma, Volkert; Henriksen, Trine; Weimann, Allan; Petersen, Morten; Andersen, Jon T.; Jimenez-Solem, Espen; Hansen, Lars J.; Henriksen, Jan Erik; Bonnema, Steen J.; de Fine Olivarius, Niels; Friis, Søren; Poulsen, Henrik E.

    2014-01-01

    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 of the RNA oxidation marker 8-oxoGuo (unadjusted hazard ratio for cancer per natural log increase in 8-oxoGuo 1.35 [95% CI, 1.01–1.81]), however, in the adjusted analyses, no significant associations between 8-oxodG or 8-oxoGuo and overall cancer were found. For site-specific cancers 8-oxodG was associated 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 ratio1.98 [95% CI, 0.95–4.10]). 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. PMID:25498965

  5. Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes

    Indian Academy of Sciences (India)

    Indraneel Mittra; Naveen Kumar Khare; Gorantla Venkata Raghuram; Rohan Chaubal; Fatema Khambatti; Deepika Gupta; Ashwini Gaikwad; Preeti Prasannan; Akshita Singh; Aishwarya Iyer; Ankita Singh; Pawan Upadhyay; Naveen Kumar Nair; Pradyumna Kumar Mishra; Amit Dutt

    2015-03-01

    Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored. We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer.

  6. 75 FR 22814 - Guidance for Industry: Nucleic Acid Testing (NAT) for Human Immunodeficiency Virus Type 1 (HIV-1...

    Science.gov (United States)

    2010-04-30

    ... (70 FR 43439), FDA announced the availability of the draft guidance of the same title. FDA received...: Nucleic Acid Testing (NAT) for Human Immunodeficiency Virus Type 1 (HIV-1) and Hepatitis C Virus (HCV... Immunodeficiency Virus Type 1 (HIV-1) and Hepatitis C Virus (HCV): Testing, Product Disposition, and Donor...

  7. Visual detection of Ebola virus using reverse transcription loop-mediated isothermal amplification combined with nucleic acid strip detection.

    Science.gov (United States)

    Xu, Changping; Wang, Hualei; Jin, Hongli; Feng, Na; Zheng, Xuexing; Cao, Zengguo; Li, Ling; Wang, Jianzhong; Yan, Feihu; Wang, Lina; Chi, Hang; Gai, Weiwei; Wang, Chong; Zhao, Yongkun; Feng, Yan; Wang, Tiecheng; Gao, Yuwei; Lu, Yiyu; Yang, Songtao; Xia, Xianzhu

    2016-05-01

    Ebola virus (species Zaire ebolavirus) (EBOV) is highly virulent in humans. The largest recorded outbreak of Ebola hemorrhagic fever in West Africa to date was caused by EBOV. Therefore, it is necessary to develop a detection method for this virus that can be easily distributed and implemented. In the current study, we developed a visual assay that can detect EBOV-associated nucleic acids. This assay combines reverse transcription loop-mediated isothermal amplification and nucleic acid strip detection (RT-LAMP-NAD). Nucleic acid amplification can be achieved in a one-step process at a constant temperature (58 °C, 35 min), and the amplified products can be visualized within 2-5 min using a nucleic acid strip detection device. The assay is capable of detecting 30 copies of artificial EBOV glycoprotein (GP) RNA and RNA encoding EBOV GP from 10(2) TCID50 recombinant viral particles per ml with high specificity. Overall, the RT-LAMP-NAD method is simple and has high sensitivity and specificity; therefore, it is especially suitable for the rapid detection of EBOV in African regions. PMID:26831931

  8. Genome Sequence of a Candidate World Health Organization Reference Strain of Zika Virus for Nucleic Acid Testing.

    Science.gov (United States)

    Trösemeier, Jan-Hendrik; Musso, Didier; Blümel, Johannes; Thézé, Julien; Pybus, Oliver G; Baylis, Sally A

    2016-01-01

    We report here the sequence of a candidate reference strain of Zika virus (ZIKV) developed on behalf of the World Health Organization (WHO). The ZIKV reference strain is intended for use in nucleic acid amplification (NAT)-based assays for the detection and quantification of ZIKV RNA. PMID:27587826

  9. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2014-04-08

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  10. Inhibition of non-templated nucleotide addition by DNA polymerases in primer extension using twisted intercalating nucleic acid modified templates

    Czech Academy of Sciences Publication Activity Database

    Güixens Gallardo, Pedro; Hocek, Michal; Perlíková, Pavla

    2016-01-01

    Roč. 26, č. 2 (2016), s. 288-291. ISSN 0960-894X R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : DNA polymerases * nucleotide addition * primer extension * oligonucleotides * twisted intercalating nucleic acid Subject RIV: CC - Organic Chemistry Impact factor: 2.420, year: 2014

  11. Nucleic-acid-programmed Ag-nanoclusters as a generic platform for visualization of latent fingerprints and exogenous substances.

    Science.gov (United States)

    Ran, Xiang; Wang, Zhenzhen; Zhang, Zhijun; Pu, Fang; Ren, Jinsong; Qu, Xiaogang

    2016-01-11

    We display a nucleic acid controlled AgNC platform for latent fingerprint visualization. The versatile emission of aptamer-modified AgNCs was regulated by the nearby DNA regions. Multi-color images for simultaneous visualization of fingerprints and exogenous components were successfully obtained. A quantitative detection strategy for exogenous substances in fingerprints was also established. PMID:26537157

  12. Biologically relevant oxidants and terminology, classification and nomenclature of oxidatively generated damage to nucleobases and 2-deoxyribose in nucleic acids

    DEFF Research Database (Denmark)

    Cadet, Jean; Loft, Steffen; Olinski, Ryszard;

    2012-01-01

    A broad scientific community is involved in investigations aimed at delineating the mechanisms of formation and cellular processing of oxidatively generated damage to nucleic acids. Perhaps as a consequence of this breadth of research expertise, there are nomenclature problems for several of the...

  13. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Ward, Thomas E.

    2016-03-22

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  14. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    Science.gov (United States)

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2013-07-23

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  15. First-principles study of physisorption of nucleic acid bases on small-diameter carbon nanotubes

    International Nuclear Information System (INIS)

    We report the results of our first-principles study based on density functional theory on the interaction of the nucleic acid base molecules adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), with a single-walled carbon nanotube (CNT). Specifically, the focus is on the physisorption of base molecules on the outer wall of a (5, 0) metallic CNT possessing one of the smallest diameters possible. Compared to the case for CNTs with large diameters, the physisorption energy is found to be reduced in the high-curvature case. The base molecules exhibit significantly different interaction strengths and the calculated binding energies follow the hierarchy G>A>T>C>U, which appears to be independent of the tube curvature. The stabilizing factor in the interaction between the base molecule and CNT is dominated by the molecular polarizability that allows a weakly attractive dispersion force to be induced between them. The present study provides an improved understanding of the role of the base sequence in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in their interactions with carbon nanotubes of varying diameters

  16. Effect of gamma-irradiation on nucleic acids, proteins, respiration and phosphatase activity of carrot callus cultures

    International Nuclear Information System (INIS)

    Callus tissue cultures were subjected to 60Co qamma irradiation at 0.5 Krad and analysed for nucleic acids, proteins, respiration rate and phosphatase activity on 0, 10, 20 and 30 days. The RNA contents and respiratory rates were enhanced as a result of irradiation. The RNA contents were reduced than their non-irradiated counterparts. The acid phosphatase activity was enhanced immediately after irradiation, declined on 10th and 20th day and more thereafter. (author)

  17. Recent developments in nucleic acid based techniques for use in rumen manipulation

    Directory of Open Access Journals (Sweden)

    Christopher McSweeney

    2009-07-01

    Full Text Available Nucleic acid-based techniques which can be used to characterise complex microbial communities without incubation are now being employed regularly in ruminant nutrition studies. Conventional culture-based methods for enumerating rumen microorganisms (bacteria, archaea, protozoa, and fungi have been superseded and are now used mainly to obtain pure isolates of novel organisms and reference strains that are required for the development and validation of the nucleic acid approaches. These reference strains are also essential for physiological studies of the lifestyle of the organisms as well as sources of genomic DNA and RNA that can be analysed for functional gene activity. The foundation of the molecular ecology techniques is 16S/18S rDNA sequence analysis which has provided a phylogenetically based classification scheme for enumeration and identification of microbial community members. The use of this marker gene in assays involving the use of single nucleic acid probes or primer sets is rapidly evolving to high throughput approaches such as microarray analysis and new generation sequencing technologies. While these analyses are very informative for determining the composition of the microbial community and monitoring changes in population size, they can only infer function based on these observations. The focus of nucleic acid research is now shifting to the functional analysis of the ecosystem which involves the measurement of functional genes and their expression in the predominant or specific members of the rumen microbial community. Functional gene studies are less developed than 16S rDNA-based analysis of community structure. Also for gene expression studies there are inherent problems involved in extracting high quality RNA from digesta, and priming cDNA synthesis from bacterial mRNA. This paper reviews nucleic acid based molecular methods which have recently been developed for studying the structure and function of rumen microbial

  18. Detection and identification of lactic acid bacteria in milk and industrial starter culture with fluorescently labeled rRNA-targeted peptide nucleic acid probes

    OpenAIRE

    Matte-Tailliez, Oriane; Quénée, Pascal; ÇIBIK, Recep; Van Opstal, Jacques; Dessevre, Fabrice; Firmesse, Olivier; Tailliez, Patrick

    2001-01-01

    Détection et identification des bactéries lactiques dans le lait et les levains industriels par des sondes "peptide nucleic acids " fluorescentes ciblant l'ARNr. Une méthode rapide et simple pour détecter et identifier les bactéries lactiques en croissance, au niveau cellulaire, dans le lait ou les levains industriels a été développée. Elle repose sur la technique d'hybridation de sondes fluorescentes à l'ARN ribosomique présent dans les cellules entières. Les "peptide nucleic acids " (PNA), ...

  19. Chemical speciation and equilibria of some nucleic acid compounds and their iron(III) complexes

    Science.gov (United States)

    Masoud, Mamdouh S.; Abd El-Kaway, Marwa Y.; Hindawy, Ahmed M.; Soayed, Amina A.

    The pH effect on electronic absorption spectra of some biologically active nucleic acid constituents have been studied at room temperature and the mechanism of ionization was explained. These compounds are of two categories (pyrimidines: [barbital; 5,5'-diethyl-barbituric acid], [SBA; 4,6-dihydroxy-2-mercapto-pyrimidin], [NBA; 5-nitro-2,4,6(1H,3H,5H)-pyrimidine trione] and [TU; 2,3-dihydro-2-thioxo-pyrimidin-4(1H)-one]) and (purines: [adenine; 6-amino purine], its [Schiff bases derived from adenine-acetylacetone; (Z)-4-(7H-purin-6-ylimino)pentan-2-one) and adenine-salicylaldehyde; 2-((7H-purin-6-ylimino) methyl) phenol] and its [Azo derived from adenine-resorcinol; 4-((7H-purin-6-yl)-diazenyl) benzene-1,3-diol]. The phenomena of tautomerization assigned different tautomers. Different spectrophotometric methods are applied to evaluate the pK's values that explained with their molecular structures. The interaction of Fe3+ with some selected pyrimidines (barbital, NBA and SBA) was explained using familiar six spectrophotometric methods. The data typified the existence of different absorbing species with the different stoichiometries 1:1, 1:2, 1:3 and 2:3. The stability constant of the complexes was computed. More approach was deduced to assign the existence of different species applying the distribution diagrams.

  20. X-ray crystal structure of N-6 adenine deoxyribose nucleic acid methyltransferase from Streptococcus pneumoniae

    Science.gov (United States)

    Tran, Phidung Hong

    X-ray diffraction by using resonant anomalous scattering has become a popular tool for solving crystal structures in the last ten years with the expanded availability of tunable synchrotron radiation for protein crystallography. Mercury atoms were used for phasing. The crystal structure of N-6 deoxyribose nucleic acid methyltransferase from Streptoccocus pneumoniae (DpnM) was solved by using the Multiple Anomalous Diffraction technique. The crystal structure reveals the formation of mercaptide between the mercury ion and the thiol group on the cysteine amino acid in a hydrophobic environment. The crystal structure contains the bound ligand, S- adenosyl-l-methionine on the surface of the concave opening. The direction of the β-strands on the beta sheets are identical to other solved methyltransferases. The highly conserved motifs, DPPY and the FxGxG, are found to be important in ligand binding and possibly in methyl group transfer. The structure has a concave cleft with an opening on the order of 30 Å that can accommodate a DNA duplex. By molecular modelling coupled to sequence alignment, two other highly conserved residues Arg21 and Gly19 are found to be important in catalysis.

  1. Complete nucleic acid sequence of Penaeus stylirostris densovirus (PstDNV) from India.

    Science.gov (United States)

    Rai, Praveen; Safeena, Muhammed P; Karunasagar, Iddya; Karunasagar, Indrani

    2011-06-01

    Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp, recently been classified as Penaeus stylirostris densovirus (PstDNV). The complete nucleic acid sequence of PstDNV from India was obtained by cloning and sequencing of different DNA fragment of the virus. The genome organisation of PstDNV revealed that there were three major coding domains: a left ORF (NS1) of 2001 bp, a mid ORF (NS2) of 1092 bp and a right ORF (VP) of 990 bp. The complete genome and amino acid sequences of three proteins viz., NS1, NS2 and VP were compared with the genomes of the virus reported from Hawaii, China and Mexico and with partial sequence available from isolates from different regions. The phylogenetic analysis of shrimp, insect and vertebrate parvovirus sequences showed that the Indian PstDNV isolate is phylogenetically more closely related to one of the three isolates from Taiwan (AY355307), and two isolates (AY362547 and AY102034) from Thailand. PMID:21402111

  2. Investigation and Manipulation of the Local Microenvironment of Spherical Nucleic Acid Nanoconjugates

    Science.gov (United States)

    Briley, William Edward

    For the past several decades, tremendous efforts have been made by many to battle cancer,one of the leading causes of death in the United States and around the world. Unfortunately, the diagnosis and treatment of many genetically-based disorders such as cancer remains very difficult to this day. This is due to the fact that current technologies are unable to adequately differentiate between healthy and diseased cells. In many cases, state-of-the-art diagnostic and therapeutics for genetic disorders rely on targeting downstream effects that may be related to, or influenced by aberrations in gene expression, rather than targeting the up- or down-regulated transcripts themselves. This type of targeting can lead to significant off-target effects, which can translate to false positives for diagnostics, and systemic toxicity for therapeutics. This thesis discusses a nanoparticle-based conjugate which aims to increase the specificity of diagnostics, therapeutics, and biological research platforms by targeting RNA transcripts directly. This nanoconjugate, known as the spherical nucleic acid (SNA) is capable of entering live cells with negligible cytotoxicity and immunogenicity, and binding onto targeted RNA transcripts. Chapter one details the properties and synthesis of the SNA, and discusses how the cell entry/transcript binding capabilities of the SNA can be translated into therapeutic and diagnostic platforms. Chapter two then moves into the therapeutic applications of the SNA, discussing a novel platform known as the Sticky-flare, which is capable of detecting and fluorescently labeling target transcripts for real time analysis. Chapter three then investigates the function of the SNA in a therapeutic application. Specifically, the route that topically applied SNAs take to penetrate through skin is elucidated, and is contextualized by comparing the penetration of SNAs with equivalent linear DNA sequences. Linear nucleic acids are typically not capable of effecting gene

  3. Imaging oncogene expression in breast cancer with receptor specific peptides and peptide nucleic acids

    International Nuclear Information System (INIS)

    Full text: This year, breast cancer (BC) will attack approximately 210, 000 and will take the lives of 40,000 women in the U.S. Standard screening with breast self-examination and mammography, recommended to minimize BC morbidity, miss 10-20% (up to 40% in young women) of breast cancer. Moreover, if an abnormality is found, an invasive diagnostic procedure is required to determine if the breast contains hyperplasia, atypia, or cancer. Approximately 80% of invasive procedures detect a benign pathology. BC cells express a gene product, cell surface receptor VPAC1, so named because the endogenous growth hormones Vasoctive Intestinal Peptide (VIP) and Pituitary Adenylate Cylcase Activating Peptide (PACAP) bind to VPAC1 receptors with high affinity. VPAC1 receptors are overexpressed on 100% of human breast cancer cells. Cyclin D1 is a key regulator of the cell cycle and overexpressed in 50% to 80% of breast cells, whereas it is low or absent in normal breast tissues. The human breast cancer cell line MCF7 displays elevated levels of CCND1 mRNA, encoding cyclin D1, and an elevated level of IGF1R mRNA, encoding insulin-like growth factor 1 receptor. We hypothesed that 99mTc or 64Cu labeled VIP analogues, or a peptide nucleic acid (PNA) chimera specific for IGFI receptor and CCND1 mRNA, will permit us to early image breast cancer by planar, SPECT or PET imaging. We synthesized, characterized and administered i.v. 99mTc-AcGly-D (Ala)-Gly-Glyaminobutanoyl- VIP (TP3654), 64Cu diaminodithiol-aminobutanoyl-VIP (TP3982), 99mTc- AcGly-D(Ala)-Gly-Gly-PNA-D(Cys-ser-lys-Cys) chimera (WT4185) and Cu-64-DOTAPNA- D(cys-ser-lys-cys) (WT4348). A 12mer, CTGGTGTTCCAT nucleic acid sequence served as the PNA and 3 or 4 mer mismatched PNAs as negative controls. Using 99mTc-TP3654 we have successfully imaged human breast cancers not detectable by current modalities. In athymic, nude mice bearing MCF-7 human breast cancer xenographs, Cu-64-TP3982 tumour uptake was 85 times greater than 99m

  4. Circular RNA oligonucleotides. Synthesis, nucleic acid binding properties, and a comparison with circular DNAs.

    Science.gov (United States)

    Wang, S; Kool, E T

    1994-06-25

    We report the synthesis and nucleic acid binding properties of two cyclic RNA oligonucleotides designed to bind single-stranded nucleic acids by pyr.pur.pyr-type triple helix formation. The circular RNAs are 34 nucleotides in size and were cyclized using a template-directed nonenzymatic ligation. To ensure isomeric 3'-5' purity in the ligation reaction, one nucleotide at the ligation site is a 2'-deoxyribose. One circle (1) is complementary to the sequence 5'-A12, and the second (2) is complementary to 5'-AAGAAAGAAAAG. Results of thermal denaturation experiments and mixing studies show that both circles bind complementary single-stranded DNA or RNA substrates by triple helix formation, in which two domains in a pyrimidine-rich circle sandwich a central purine-rich substrate. The affinities of these circles with their purine complements are much higher than the affinities of either the linear precursors or simple Watson-Crick DNA complements. For example, circle 1 binds rA12 (pH 7.0, 10 mM MgCl2, 100 mM NaCl) with a Tm of 48 degrees C and a Kd (37 degrees C) of 4.1 x 10(-9) M, while the linear precursor of the circle binds with a Tm of 34 degrees C and a Kd of 1.2 x 10(-6) M. The complexes of circle 2 are pH-dependent, as expected for triple helical complexes involving C(+)G.C triads, and mixing plots for both circles reveal one-to-one stoichiometry of binding either to RNA or DNA substrates. Comparison of circular RNAs with previously synthesized circular DNA oligonucleotides of the same sequence reveals similar behavior in the binding of DNA, but strikingly different behavior in the binding of RNA. The cyclic DNAs show high DNA-binding selectivity, giving relatively weaker duplex-type binding with complementary RNAs. The relative order of thermodynamic stability for the four types of triplex studied here is found to be DDD > RRR > RDR > DRD. The results are discussed in the context of recent reports of strong triplex dependence on RNA versus DNA backbones. Triplex

  5. The nucleic acid precursors can enhance the intestinal crypt survival in mice after 980 cGy abdominal γ-irradiation

    International Nuclear Information System (INIS)

    The effect of a single post-irradiation intestinal lumen dilatation injection of one of mononucleotides, nucleosides or nucleic acid bases on intestinal crypt survival after 980 cGy abdominally γ-irradiated mice has been studied. The results showed that any one of these nucleic acid components can produce the same enhancing effect of crypt survival as that did by polymerized calf thymus DNA. This fact suggests that the radio restorative effect of exogenous nucleic acids on the intestinal crypt cells depends not upon the action exerted by their highly polymerized state, but rather by their various enzymatic degradation products

  6. CHEMOTHERAPEUTIC POLYMERS ⅩⅩⅢ SYNTHESIS AND ANTITUMOR ACTIVITY OF POLYPHOSPHATES CONTAINING BOTH NUCLEIC ACID BASE AND PHOSPHONOACETIC ACID ETHYL ESTER

    Institute of Scientific and Technical Information of China (English)

    ZHUO Renxi; LIU Zhenghua; LI Li

    1989-01-01

    Eight new polyphosphates containing both nucleic acid base and phosphonoacetic acid ethyl ester were synthesized by the polycondensation of P, P- dichloride of phosphonoacetic acid ethyl ester with 1, 3-dihydroxyalkyl - 5 - fluorouracil, 1,3 - dihydroxyalkyl - uracil and 1, 3 - dihydroxyalkylthymine. These polyphosphates were tested against Ehrlich Ascites Carcinoma in mice. Polymer Ⅱa and Ⅱc exhibited excellent antitumor activity. Ⅱc also showed lower toxicity.

  7. On the role of mercury in the non-covalent stabilisation of consecutive U-HgII-U metal-mediated nucleic acid base pairs: metallophilic attraction enters the world of nucleic acids

    Czech Academy of Sciences Publication Activity Database

    Benda, Ladislav; Straka, Michal; Yoshiyuki, T.; Sychrovský, Vladimír

    2011-01-01

    Roč. 13, č. 1 (2011), s. 100-103. ISSN 1463-9076 R&D Projects: GA ČR GA203/09/2037; GA ČR GAP205/10/0228 Grant ostatní: European Reintegration Grant(XE) 230955 Institutional research plan: CEZ:AV0Z40550506 Keywords : metallophilic attraction * nucleic acid * mercury Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.573, year: 2011

  8. Comparative distribution study of C labelled amino acids, glucose-analogue and precursor of nucleic acid, as tumor seeking agents

    Energy Technology Data Exchange (ETDEWEB)

    Shiba, Kazuhiro; Mori, Hirofumi; Hisada, Kinichi

    1984-08-01

    As tumor-seeking agents, glucose analogues, natural amino acids, synthetic nonmetabolized amino acids, and precursor of nucleic acids, etc., labeled with positron emitter, such as C and YF have been recently investigated. However, there are very few reports concerning comparative study of tumor uptake and tissue distribution of these agents. This preliminary paper describes comparative distribution and whole-body autoradiography of these agents. UC labeled deoxy-2-fluoro-D-glucose (FDG), L-, DL-leucine, 1-aminocyclopentane carboxylic acid (ACPC), -amino isobutyric acid ( -AIB), and thymidine were intravenously injected through tail vein into separate groups of the experimental animals. As the experimental animals, the mice with Ehrlich tumor and the rats with Hepatoma AH109A were used. Within 30 min after injection, FDG had the highest tumor uptake and tumor to tissue ratios, although FDG was inferior to ACPC and thymidine in related to tumor to heart, lung and brain ratios. However, the time course study indicated that tumor uptake of ACPC, -AIB and D-leucine increased with time, whereas those of other agents decreased with time or reached a plateau. Thus, at 120 min after injection, ACPC had the highest tumor uptake and tumor to tissue ratios, although ACPC was inferior to FDG in related to tumor to blood, liver and pancreas ratios. Autoradiogram of ACPC showed very clear tumor image as well as that of FDG. The above data suggest that synthetic nonmetabolized amino acids, such as ACPC may be promising as tumor-seeking agents, when used with a single photon emission computed tomography, while glucose analogue such as FDG, are the best tumor-seeking agent, when used with a positron emission computed tomography. (author).

  9. Nucleic Acids and Protein Metabolism of Bone Marrow Cells Studied by Means of Tritiumlabelled Precursors

    International Nuclear Information System (INIS)

    The advantages of the use of tritium-labelled compounds in radioautographic technique are discussed. Tritium electrons have a maximal energy of 0.018 MeV, corresponding to about 1μm range in a photographic emulsion, and consequently they allow the highest possible resolution at a cellular and subcellular level. This is particularly useful for studying metabolic phenomena of tissues which are composed, as in the case of bone marrow, of different cellular types at various stages of differentiation. This technique has been used for investigating nucleic acids and protein metabolism of normal and leukaemic bone marrow cells. DNA metabolism has been studied utilizing a specific precursor, H3-thymidine. Some significant differences of the percentages of labelled cells have been detected by comparing the normal and leukaemic elements belonging to the same stage of maturation. In acute leukaemia cells, particularly, a strikingly lower incorporation of thymidine was found and these results have been taken as evidence of a decreased proliferative capacity of these cells, as compared to normal myeloblasts. With the same technique, RNA and protein metabolism have been investigated utilizing H3- uridine, H3-leucine and H3-phenylalanine as precursors. The existence of a strict interrelationship between RNA and protein metabolism is now fully accepted in cellular biology. The existence of a constant ratio between uridine and amino acids incorporation has also been demonstrated in normal bone marrow cells. In acute leukaemia cells the incorporation of RNA and protein precursors, although different from case to case, is constantly and significantly lower. Furthermore, the ratio between uridine and amino acids incorporation is constantly altered in these cells. The lower RNA and protein metabolism and its dissociation in acute leukaemia cells is discussed in relation to the well-known maturation defect of these cells. (author)

  10. Proteomic Retrieval from Nucleic Acid Depleted Space-Flown Human Cells

    Science.gov (United States)

    Hammond, D. K.; Elliott, T. F.; Holubec, K.; Baker, T. L.; Allen, P. L.; Hammond, T. G.; Love, J. E.

    2006-01-01

    Compared to experiments utilizing humans in microgravity, cell-based approaches to questions about subsystems of the human system afford multiple advantages, such as crew safety and the ability to achieve statistical significance. To maximize the science return from flight samples, an optimized method was developed to recover protein from samples depleted of nucleic acid. This technique allows multiple analyses on a single cellular sample and when applied to future cellular investigations could accelerate solutions to significant biomedical barriers to human space exploration. Cell cultures grown in American Fluoroseal bags were treated with an RNA stabilizing agent (RNAlater - Ambion), which enabled both RNA and immunoreactive protein analyses. RNA was purified using an RNAqueous(registered TradeMark) kit (Ambion) and the remaining RNA free supernatant was precipitated with 5% trichloroacetic acid. The precipitate was dissolved in SDS running buffer and tested for protein content using a bicinchoninic acid assay (1) (Sigma). Equal loads of protein were placed on SDS-PAGE gels and either stained with CyproOrange (Amersham) or transferred using Western Blotting techniques (2,3,4). Protein recovered from RNAlater-treated cells and stained with protein stain, was measured using Imagequant volume measurements for rectangles of equal size. BSA treated in this way gave quantitative data over the protein range used (Fig 1). Human renal cortical epithelial (HRCE) cells (5,6,7) grown onboard the International Space Station (ISS) during Increment 3 and in ground control cultures exhibited similar immunoreactivity profiles for antibodies to the Vitamin D receptor (VDR) (Fig 2), the beta isoform of protein kinase C (PKC ) (Fig 3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Fig 4). Parallel immunohistochemical studies on formalin-fixed flight and ground control cultures also showed positive immunostaining for VDR and other biomarkers (Fig 5). These results are

  11. Erythrocyte cycle of Plasmodium falciparum. Synthesis of nucleic acids and proteins

    International Nuclear Information System (INIS)

    In vitro cultures of Plasmodium falciparum (FCR-7 strain) have been synchronized by starting with a culture parasitaemia of about 10%, of which at least half consisted of trophozoites and schizonts, and sedimentation in physiogel at 37 deg. C for 30 min. Unparasitized red cells and ring-form parasites from rouleaux and sediment. Trophozoites and schizonts remain in suspension. After washing, these latter are used to inoculate fresh unparasitized red cells, thus starting a synchronized culture. This, although not perfect, is satisfactory, yielding clear separation of the peaks of the various morphological forms determined by differential counting of smears made at the end of each pulse-labelling period. To label nucleic acids aliquots of this culture received 2-h pulses of 3H-hypoxanthine. After treating one with ribonuclease, the levels of incorporation into trichloroacetic acid (TCA) precipitates were determined for each aliquot. The procedure for labelling proteins was the same except that 35S-methionine replaced hypoxanthine and there was no ribonuclease treatment, so that the duplicates were identical. Serum was omitted from all pulse-labelling incubations. The data obtained agree with earlier reports on P. knowlesi in that the rate of nuclei acid synthesis is highest during the trophozoite stage and falls off in schizogony. It is unlikely that the low incorporation into the ring forms is due to the differences in permeability, since red cells are highly permeable to hypoxanthine. Most of the protein synthesis occurs during the trophozoite stage, with a second burst in the schizonts. It is low but still significant in the ring forms. Polyacrylamide gel electrophoresis followed by fluorography showed a few bands specific to each stage of maturation, in contradiction to some earlier reports. One of these, specific to the schizonts, has a molecular weight (by electrophoretic mobility) of 50,000 and it may be identical with the histidine-rich protein described by

  12. Orientation Preferences of Backbone Secondary Amide Functional Groups in Peptide Nucleic Acid Complexes: Quantum Chemical Calculations Reveal an Intrinsic Preference of Cationic D-Amino Acid-Based Chiral PNA Analogues for the P-form

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Jeremy C [ORNL; Topham, Christopher [University of Heidelberg

    2007-01-01

    Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like base pair stacking. Quantum chemical calculations on the binding of a water molecule to an O2 base atom in glycine-based PNA thymine dimers indicate that junctions modeled with P-form backbone conformations are lower in energy than a dimer comprising the predominant conformation observed in A-like helices. It is further shown in model systems that PNA analogs based on D-lysine are better able to preorganize in a conformation exclusive to P-form helices than is glycine-based PNA. An intrinsic preference for this conformation is also exhibited by positively charged chiral PNA dimers carrying 3-amino-D-alanine or 4-aza-D-leucine residue units that provide for additional rigidity by side-chain hydrogen bonding to the backbone carbonyl oxygen. Structural modifications stabilizing P-form helices may obviate the need for large heterocycles to target DNA pyrimidine bases via PNADNA-PNA triplex formation. Quantum chemical modeling methods are used to propose candidate PNA Hoogsteen strand designs.

  13. Orientation preferences of backbone secondary amide functional groups in peptide nucleic acid complexes: quantum chemical calculations reveal an intrinsic preference of cationic D-amino acid-based chiral PNA analogues for the P-form.

    Science.gov (United States)

    Topham, Christopher M; Smith, Jeremy C

    2007-02-01

    Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like basepair stacking. Quantum chemical calculations on the binding of a water molecule to an O2 base atom in glycine-based PNA thymine dimers indicate that junctions modeled with P-form backbone conformations are lower in energy than a dimer comprising the predominant conformation observed in A-like helices. It is further shown in model systems that PNA analogs based on D-lysine are better able to preorganize in a conformation exclusive to P-form helices than is glycine-based PNA. An intrinsic preference for this conformation is also exhibited by positively charged chiral PNA dimers carrying 3-amino-D-alanine or 4-aza-D-leucine residue units that provide for additional rigidity by side-chain hydrogen bonding to the backbone carbonyl oxygen. Structural modifications stabilizing P-form helices may obviate the need for large heterocycles to target DNA pyrimidine bases via PNA.DNA-PNA triplex formation. Quantum chemical modeling methods are used to propose candidate PNA Hoogsteen strand designs. PMID:17071666

  14. Nucleic acid-amplification testing for hepatitis B in cornea donors.

    Science.gov (United States)

    Fornés, Maria Gema; Jiménez, Maria Angustias; Eisman, Marcela; Gómez Villagrán, Jose Luis; Villalba, Rafael

    2016-06-01

    Careful donor selection and implementation of tests of appropriate sensitivity and specificity are of paramount importance for minimizing the risk of transmitting infectious diseases from donors to corneal allograft recipients. Reported cases of viral transmission with corneal grafts are very unusual. Nevertheless potential virus transmission through the engraftment cannot be ruled out. According to European Guideline 2006/17/EC, screening for antibodies for Hepatitis B core antigen (anti HBc) is mandatory, and when this test is positive, some criteria must be established before using corneas. Despite the continuous progress in screening tests, donors carrying an occult hepatitis B infection (OBI) can cause transplant-transmitted hepatitis B. To date, Nucleic Acid Testing (NAT) is not an obligatory assay in corneal tissue setting neither in our country nor in the rest of European countries. Herein, we report three cornea donors that were rejected with the diagnosis of OBI through the testing of sensitive NAT and the serological profile of Hepatitis B virus. The aim of this report is to emphasize the need to include NAT in new reviews of EU Tissues and Cells Directives in order to increase level of security in tissue donation as well as not to reject a high number of donors with isolated profile of anti HBc in geographical areas with high prevalence of Hepatitis B, that could be rejected without a true criterion of Hepatitis B infection. PMID:26685699

  15. In vivo encapsulation of nucleic acids using an engineered nonviral protein capsid.

    Science.gov (United States)

    Lilavivat, Seth; Sardar, Debosmita; Jana, Subrata; Thomas, Geoffrey C; Woycechowsky, Kenneth J

    2012-08-15

    In Nature, protein capsids function as molecular containers for a wide variety of molecular cargoes. Such containers have great potential for applications in nanotechnology, which often require encapsulation of non-native guest molecules. Charge complementarity represents a potentially powerful strategy for engineering novel encapsulation systems. In an effort to explore the generality of this approach, we engineered a nonviral, 60-subunit capsid, lumazine synthase from Aquifex aeolicus (AaLS), to act as a container for nucleic acid. Four mutations were introduced per subunit to increase the positive charge at the inner surface of the capsid. Characterization of the mutant (AaLS-pos) revealed that the positive charges lead to the uptake of cellular RNA during production and assembly of the capsid in vivo. Surprisingly, AaLS-pos capsids were found to be enriched with RNA molecules approximately 200-350 bases in length, suggesting that this simple charge complementarity approach to RNA encapsulation leads to both high affinity and a degree of selectivity. The ability to control loading of RNA by tuning the charge at the inner surface of a protein capsid could illuminate aspects of genome recognition by viruses and pave the way for the development of improved RNA delivery systems. PMID:22827162

  16. Enzyme-free detection and quantification of double-stranded nucleic acids.

    Science.gov (United States)

    Feuillie, Cécile; Merheb, Maxime Mohamad; Gillet, Benjamin; Montagnac, Gilles; Hänni, Catherine; Daniel, Isabelle

    2012-08-01

    We have developed a fully enzyme-free SERRS hybridization assay for specific detection of double-stranded DNA sequences. Although all DNA detection methods ranging from PCR to high-throughput sequencing rely on enzymes, this method is unique for being totally non-enzymatic. The efficiency of enzymatic processes is affected by alterations, modifications, and/or quality of DNA. For instance, a limitation of most DNA polymerases is their inability to process DNA damaged by blocking lesions. As a result, enzymatic amplification and sequencing of degraded DNA often fail. In this study we succeeded in detecting and quantifying, within a mixture, relative amounts of closely related double-stranded DNA sequences from Rupicapra rupicapra (chamois) and Capra hircus (goat). The non-enzymatic SERRS assay presented here is the corner stone of a promising approach to overcome the failure of DNA polymerase when DNA is too degraded or when the concentration of polymerase inhibitors is too high. It is the first time double-stranded DNA has been detected with a truly non-enzymatic SERRS-based method. This non-enzymatic, inexpensive, rapid assay is therefore a breakthrough in nucleic acid detection. PMID:22695500

  17. Fluorescent Probes for Nucleic Acid Visualization in Fixed and Live Cells

    Directory of Open Access Journals (Sweden)

    Alexandre S. Boutorine

    2013-12-01

    Full Text Available This review analyses the literature concerning non-fluorescent and fluorescent probes for nucleic acid imaging in fixed and living cells from the point of view of their suitability for imaging intracellular native RNA and DNA. Attention is mainly paid to fluorescent probes for fluorescence microscopy imaging. Requirements for the target-binding part and the fluorophore making up the probe are formulated. In the case of native double-stranded DNA, structure-specific and sequence-specific probes are discussed. Among the latest, three classes of dsDNA-targeting molecules are described: (i sequence-specific peptides and proteins; (ii triplex-forming oligonucleotides and (iii polyamide oligo(N-methylpyrrole/N-methylimidazole minor groove binders. Polyamides seem to be the most promising targeting agents for fluorescent probe design, however, some technical problems remain to be solved, such as the relatively low sequence specificity and the high background fluorescence inside the cells. Several examples of fluorescent probe applications for DNA imaging in fixed and living cells are cited. In the case of intracellular RNA, only modified oligonucleotides can provide such sequence-specific imaging. Several approaches for designing fluorescent probes are considered: linear fluorescent probes based on modified oligonucleotide analogs, molecular beacons, binary fluorescent probes and template-directed reactions with fluorescence probe formation, FRET donor-acceptor pairs, pyrene excimers, aptamers and others. The suitability of all these methods for living cell applications is discussed.

  18. Excretion and detection of SARS coronavirus and its nucleic acid from digestive system

    Institute of Scientific and Technical Information of China (English)

    Xin-Wei Wang; Xiao-Ming Wu; Wen-Jun Xiao; Xiu-Mei Zhu; Chang-Qing Gu; Jing Yin; Wei Wei; Wei Yao; Chao Liu; Jian-Feng Li; Guo-Rong Ou; Jin-Song Li; Min-Nian Wang; Tong-Yu Fang; Gui-Jie Wang; Yao-Hui Qiu; Huai-Huan Wu; Fu-Huan Chao; Jun-Wen Li; Ting-Kai Guo; Bei Zhen; Qing-Xin Kong; Bin Yi; Zhong Li; Nong Song; Min Jin

    2005-01-01

    AIM: To study whether severe acute respiratory syndrome coronavirus (SARS-CoV) could be excreted from digestive system.METHODS: Cell culture and semi-nested RT-PCR were used to detect SARS-CoV and its RNA from 21 stool and urine samples, and a kind of electropositive filter media particles was used to concentrate the virus in 10 sewage samples from two hospitals receiving SAPS patients in Beijing in China.RESULTS: It was demonstrated that there was no live SARS-CoV in all samples collected, but the RNA of SARS-CoV could be detected in seven stool samples from SARS patients with any one of the symptoms of fever, malaise,cough, or dyspnea, in 10 sewage samples before disinfection and 3 samples after disinfection from the two hospitals.The RNA could not be detected in urine and stool samples from patients recovered from SARS.CONCLUSION: Nucleic acid of SARS-CoV can be excreted through the stool of patients into sewage system, and the possibility of SARS-CoV transmitting through digestive system cannot be excluded.

  19. Efficient isolation of high quality nucleic acids from different tissues of Taxus baccata L.

    Science.gov (United States)

    Abbasi Kejani, Abolghasem; Hosseini Tafreshi, Sayed Ali; Khayyam Nekouei, Sayed Mojtaba; Mofid, Mohammad Reza

    2010-02-01

    Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and beta-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A(260)/A(280) and A(260)/A(230) ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100-300 microg/g leaf and stem tissue and total RNA with an average yield of 20-30 microg/g cell culture and 80-100 microg/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively. PMID:19578976

  20. Intrinsic nucleic acid dynamics modulates HIV-1 nucleocapsid protein binding to its targets.

    Science.gov (United States)

    Bazzi, Ali; Zargarian, Loussiné; Chaminade, Françoise; De Rocquigny, Hugues; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2012-01-01

    HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem. To further understand how this site was selected among several potential binding sites containing unpaired guanines, we probed the intrinsic dynamics of mini-cTAR using (13)C relaxation measurements. Results of spin relaxation time measurements have been analyzed using the model-free formalism and completed by dispersion relaxation measurements. Our data indicate that the preferentially recognized guanine in the lower part of the stem is exempt of conformational exchange and highly mobile. In contrast, the unrecognized unpaired guanines of mini-cTAR are involved in conformational exchange, probably related to transient base-pairs. These findings support the notion that NC preferentially recognizes unpaired guanines exhibiting a high degree of mobility. The ability of NC to discriminate between close sequences through their dynamic properties contributes to understanding how NC recognizes specific sites within the HIV genome. PMID:22745685