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1

Nucleic acid isolation process  

DOEpatents

A method is provided for isolating DNA from eukaryotic cell and flow sorted chromosomes. When DNA is removed from chromosome and cell structure, detergent and proteolytic digestion products remain with the DNA. These products can be removed with organic extraction, but the process steps associated with organic extraction reduce the size of DNA fragments available for experimental use. The present process removes the waste products by dialyzing a solution containing the DNA against a solution containing polyethylene glycol (PEG). The waste products dialyze into the PEG leaving isolated DNA. The remaining DNA has been prepared with fragments containing more than 160 kb. The isolated DNA has been used in conventional protocols without affect on the protocol.

Longmire, Jonathan L. (Los Alamos, NM); Lewis, Annette K. (La Jolla, CA); Hildebrand, Carl E. (Los Alamos, NM)

1990-01-01

2

Nucleic acid isolation  

DOEpatents

A method is provided for isolating DNA from eukaryotic cell and flow sorted chromosomes. When DNA is removed from chromosome and cell structure, detergent and proteolytic digestion products remain with the DNA. These products can be removed with organic extraction, but the process steps associated with organic extraction reduces the size of DNA fragments available for experimental use. The present process removes the waste products by dialyzing a solution containing the DNA against a solution containing polyethylene glycol (PEG). The waste products dialyze into the PEG leaving isolated DNA. The remaining DNA has been prepared with fragments containing more than 160 kb. The isolated DNA has been used in conventional protocols without effect on the protocol.

Longmire, J.L.; Lewis, A.K.; Hildebrand, C.E.

1988-01-21

3

Method for nucleic acid isolation using supercritical fluids  

DOEpatents

A method for detecting the presence of a microorganism in an environmental sample involves contacting the sample with a supercritical fluid to isolate nucleic acid from the microorganism, then detecting the presence of a particular sequence within the isolated nucleic acid. The nucleic acid may optionally be subjected to further purification.

Nivens, David E. (11912 Kingsgate Rd., Knoxville, TN 37911); Applegate, Bruce M. (3700 Sutherland Ave. #Q2, Knoxville, TN 37911)

1999-01-01

4

Silicon Dioxide Thin Film Mediated Single Cell Nucleic Acid Isolation  

PubMed Central

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

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

2013-01-01

5

Silicon dioxide thin film mediated single cell nucleic acid isolation.  

PubMed

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

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

2013-01-01

6

Rapid and efficient isolation of high quality nucleic acids from plant tissues rich in polyphenols and polysaccharides.  

PubMed

Isolation of high quality nucleic acids from plant tissues rich in polysaccharides and polyphenols is often difficult. The presence of these substances can affect the quality and/or quantity of the nucleic acids isolated. Here, we describe a rapid and efficient nucleic acids extraction protocol that in contrast to other methods tested, effectively purify high quality nucleic acids from plant tissues rich in polysaccharides and polyphenolic compounds such as different grape tissues and fruit tissue of fruit trees. The nucleic acids isolated with this protocol were successfully used for many functional genomic based experiments including polymerase chain reaction, reverse transcription polymerase chain reaction (RT-PCR), cloning, and semiquantitative RT-PCR. PMID:21302150

Japelaghi, Reza Heidari; Haddad, Raheem; Garoosi, Ghasem-Ali

2011-10-01

7

An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids.  

PubMed

A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid-based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids. PMID:20401537

Chen, Dafeng; Mauk, Michael; Qiu, Xianbo; Liu, Changchun; Kim, Jitae; Ramprasad, Sudhir; Ongagna, Serge; Abrams, William R; Malamud, Daniel; Corstjens, Paul L A M; Bau, Haim H

2010-08-01

8

An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids  

PubMed Central

A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid—based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids. PMID:20401537

Chen, Dafeng; Mauk, Michael; Qiu, Xianbo; Liu, Changchun; Kim, Jitae; Ramprasad, Sudhir; Ongagna, Serge; Abrams, William R.; Malamud, Daniel; Corstjens, Paul L. A. M.

2010-01-01

9

Nucleic Acid Isolation and Enrichment on a Microchip  

PubMed Central

This paper presents a microchip that isolates and enriches target-binding single-stranded DNA (ssDNA) from a randomized DNA mixture using a combination of solid-phase extraction and electrophoresis. Strands of ssDNA in a randomized mixture are captured via specific binding onto target-functionalized microbeads in a microchamber. The strands are further separated from impurities and enriched on-chip via electrophoresis. The microchip consists of two microchambers that are connected by a channel filled with agarose gel. In the isolation chamber, beads functionalized with human immunoglobulin E (IgE) are retained by a weir structure. An integrated heater elevates the temperature in the chamber to elute desired ssDNA from the beads, and electrophoretic transport of the DNA through the gel to the second chamber is accomplished by applying an electric potential difference between the two chambers. Experimental results show that ssDNA expressing binding affinity to IgE was captured and enriched from a sample of ssDNA with random sequences, demonstrating the potential of the microchip to enhance the sensitivity of ssDNA detection methods in dilute and complex biological samples. PMID:24729660

Kim, Jinho; Hilton, John P.; Yang, Kyung A.; Pei, Renjun; Stojanovic, Milan; Lin, Qiao

2014-01-01

10

Isolation of nucleic acids and cultures from fossil ice and permafrost  

Microsoft Academic Search

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 microbial nucleic acids obtained from ice- and permafrost cores from hundreds of thousands to millions

Eske Willerslev; Anders J. Hansen; Hendrik N. Poinar

2004-01-01

11

Microfluidic platform for isolating nucleic acid targets using sequence specific hybridization  

PubMed Central

The separation of target nucleic acid sequences from biological samples has emerged as a significant process in today's diagnostics and detection strategies. In addition to the possible clinical applications, the fundamental understanding of target and sequence specific hybridization on surface modified magnetic beads is of high value. In this paper, we describe a novel microfluidic platform that utilizes a mobile magnetic field in static microfluidic channels, where single stranded DNA (ssDNA) molecules are isolated via nucleic acid hybridization. We first established efficient isolation of biotinylated capture probe (BP) using streptavidin-coated magnetic beads. Subsequently, we investigated the hybridization of target ssDNA with BP bound to beads and explained these hybridization kinetics using a dual-species kinetic model. The number of hybridized target ssDNA molecules was determined to be about 6.5 times less than that of BP on the bead surface, due to steric hindrance effects. The hybridization of target ssDNA with non-complementary BP bound to bead was also examined, and non-specific hybridization was found to be insignificant. Finally, we demonstrated highly efficient capture and isolation of target ssDNA in the presence of non-target ssDNA, where as low as 1% target ssDNA can be detected from mixture. The microfluidic method described in this paper is significantly relevant and is broadly applicable, especially towards point-of-care biological diagnostic platforms that require binding and separation of known target biomolecules, such as RNA, ssDNA, or protein. PMID:24404041

Wang, Jingjing; Morabito, Kenneth; Tang, Jay X.; Tripathi, Anubhav

2013-01-01

12

Portable nucleic acid thermocyclers.  

PubMed

A nucleic acid thermal cycler is considered to be portable if it is under ten pounds, easily carried by one individual, and battery powered. Nucleic acid amplification includes both polymerase chain reaction (e.g. PCR, RT-PCR) and isothermal amplification (e.g. RPA, HDA, LAMP, NASBA, RCA, ICAN, SMART, SDA). There are valuable applications for portable nucleic acid thermocyclers in fields that include clinical diagnostics, biothreat detection, and veterinary testing. A system that is portable allows for the distributed detection of targets at the point of care and a reduction of the time from sample to answer. The designer of a portable nucleic acid thermocycler must carefully consider both thermal control and the detection of amplification. In addition to thermal control and detection, the designer may consider the integration of a sample preparation subsystem with the nucleic acid thermocycler. There are a variety of technologies that can achieve accurate thermal control and the detection of nucleic acid amplification. Important evaluation criteria for each technology include maturity, power requirements, cost, sensitivity, speed, and manufacturability. Ultimately the needs of a particular market will lead to user requirements that drive the decision between available technologies. PMID:24030680

Almassian, David R; Cockrell, Lisa M; Nelson, William M

2013-11-21

13

Liposomal spherical nucleic acids.  

PubMed

A novel class of metal-free spherical nucleic acid nanostructures was synthesized from readily available starting components. These particles consist of 30 nm liposomal cores, composed of an FDA-approved 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid monomer. The surface of the liposomes was functionalized with DNA strands modified with a tocopherol tail that intercalates into the phospholipid layer of the liposomal core via hydrophobic interactions. The spherical nucleic acid architecture not only stabilizes these constructs but also facilitates cellular internalization and gene regulation in SKOV-3 cells. PMID:24983505

Banga, Resham J; Chernyak, Natalia; Narayan, Suguna P; Nguyen, SonBinh T; Mirkin, Chad A

2014-07-16

14

Polyvalent nucleic acid nanostructures.  

PubMed

Polyvalent oligonucleotide-nanoparticle conjugates possess several unique emergent properties, including enhanced cellular uptake, high antisense bioactivity, and nuclease resistance, which hypothetically originate from the dense packing and orientation of oligonucleotides on the surface of the nanoparticle. In this Communication, we describe a new class of polyvalent nucleic acid nanostructures (PNANs), which are comprised of only cross-linked and oriented nucleic acids. We demonstrate that these particles are capable of effecting high cellular uptake and gene regulation without the need of a cationic polymer co-carrier. The PNANs also exhibit cooperative binding behavior and nuclease resistance properties. PMID:21630678

Cutler, Joshua I; Zhang, Ke; Zheng, Dan; Auyeung, Evelyn; Prigodich, Andrew E; Mirkin, Chad A

2011-06-22

15

Polyvalent Nucleic Acid Nanostructures  

PubMed Central

Polyvalent oligonucleotide-nanoparticle conjugates possess several unique emergent properties including enhanced cellular uptake, high antisense bioactivity, and nuclease resistance, which hypothetically originate from the dense packing and orientation of oligonucleotides on the surface of the nanoparticle. In this communication, we describe a new class of polyvalent nucleic acid nanostructures (PNANs), which comprise only crosslinked and oriented nucleic acids. We demonstrate that these particles are capable of effecting high cellular uptake and gene regulation without the need of a cationic polymer co-carrier. The PNANs also exhibit cooperative binding behavior and nuclease resistance properties. PMID:21630678

Cutler, Joshua I.; Zhang, Ke; Zheng, Dan; Auyeung, Evelyn; Prigodich, Andrew E.

2011-01-01

16

The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?  

PubMed

Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase "blocks" during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims. PMID:17579711

Gilbert, M Thomas P; Haselkorn, Tamara; Bunce, Michael; Sanchez, Juan J; Lucas, Sebastian B; Jewell, Laurence D; Van Marck, Eric; Worobey, Michael

2007-01-01

17

The Isolation of Nucleic Acids from Fixed, Paraffin-Embedded Tissues-Which Methods Are Useful When?  

PubMed Central

Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase “blocks” during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims. PMID:17579711

Gilbert, M. Thomas P.; Haselkorn, Tamara; Bunce, Michael; Sanchez, Juan J.; Lucas, Sebastian B.; Jewell, Laurence D.; Marck, Eric Van; Worobey, Michael

2007-01-01

18

Method and Apparatus for Automated Isolation of Nucleic Acids from Small Cell Samples  

NASA Technical Reports Server (NTRS)

RNA isolation is a ubiquitous need, driven by current emphasis on microarrays and miniaturization. With commercial systems requiring 100,000 to 1,000,000 cells for successful isolation, there is a growing need for a small-footprint, easy-to-use device that can harvest nucleic acids from much smaller cell samples (1,000 to 10,000 cells). The process of extraction of RNA from cell cultures is a complex, multi-step one, and requires timed, asynchronous operations with multiple reagents/buffers. An added complexity is the fragility of RNA (subject to degradation) and its reactivity to surface. A novel, microfluidics-based, integrated cartridge has been developed that can fully automate the complex process of RNA isolation (lyse, capture, and elute RNA) from small cell culture samples. On-cartridge cell lysis is achieved using either reagents or high-strength electric fields made possible by the miniaturized format. Traditionally, silica-based, porous-membrane formats have been used for RNA capture, requiring slow perfusion for effective capture. In this design, high efficiency capture/elution are achieved using a microsphere-based "microfluidized" format. Electrokinetic phenomena are harnessed to actively mix microspheres with the cell lysate and capture/elution buffer, providing important advantages in extraction efficiency, processing time, and operational flexibility. Successful RNA isolation was demonstrated using both suspension (HL-60) and adherent (BHK-21) cells. Novel features associated with this development are twofold. First, novel designs that execute needed processes with improved speed and efficiency were developed. These primarily encompass electric-field-driven lysis of cells. The configurations include electrode-containing constructs, or an "electrode-less" chip design, which is easy to fabricate and mitigates fouling at the electrode surface; and the "fluidized" extraction format based on electrokinetically assisted mixing and contacting of microbeads in a shape-optimized chamber. A secondary proprietary feature is in the particular layout integrating these components to perform the desired operation of RNA isolation. Apart from a novel functional capability, advantages of the innovation include reduced or eliminated use of toxic reagents, and operator-independent extraction of RNA.

Sundaram, Shivshankar; Prabhakarpandian, Balabhaskar; Pant, Kapil; Wang, Yi

2014-01-01

19

Shaping up nucleic acid computation  

PubMed Central

Summary of recent advances (abstract) Nucleic acid-based nanotechnology has always been perceived as novel, but has begun to move from theoretical demonstrations to practical applications. In particular, the large address spaces available to nucleic acids can be exploited to encode algorithms and/or act as circuits, and thereby process molecular information. In this review we revisit several milestones in the field of nucleic acid-based computation, but also highlight how the prospects for nucleic acid computation go beyond just a large address space. Functional nucleic acid elements (aptamers, ribozymes, and deoxyribozymes) can serve as inputs and outputs to the environment, and can act as logical elements. Into the future, the chemical dynamics of nucleic acids may prove as useful as hybridization for computation. PMID:20538451

Chen, Xi

2010-01-01

20

Nucleic Acid Detection Methods  

DOEpatents

The invention relates to methods for rapidly determining the sequence and/or length a target sequence. The target sequence may be a series of known or unknown repeat sequences which are hybridized to an array of probes. The hybridized array is digested with a single-strand nuclease and free 3'-hydroxyl groups extended with a nucleic acid polymerase. Nuclease cleaved heteroduplexes can be easily distinguish from nuclease uncleaved heteroduplexes by differential labeling. Probes and target can be differentially labeled with detectable labels. Matched target can be detected by cleaving resulting loops from the hybridized target and creating free 3-hydroxyl groups. These groups are recognized and extended by polymerases added into the reaction system which also adds or releases one label into solution. Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders. Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated.

Smith, Cassandra L. (Boston, MA); Yaar, Ron (Brookline, MA); Szafranski, Przemyslaw (Boston, MA); Cantor, Charles R. (Boston, MA)

1998-05-19

21

Nucleic acid detection methods  

DOEpatents

The invention relates to methods for rapidly determining the sequence and/or length a target sequence. The target sequence may be a series of known or unknown repeat sequences which are hybridized to an array of probes. The hybridized array is digested with a single-strand nuclease and free 3{prime}-hydroxyl groups extended with a nucleic acid polymerase. Nuclease cleaved heteroduplexes can be easily distinguish from nuclease uncleaved heteroduplexes by differential labeling. Probes and target can be differentially labeled with detectable labels. Matched target can be detected by cleaving resulting loops from the hybridized target and creating free 3-hydroxyl groups. These groups are recognized and extended by polymerases added into the reaction system which also adds or releases one label into solution. Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders. Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated. 18 figs.

Smith, C.L.; Yaar, R.; Szafranski, P.; Cantor, C.R.

1998-05-19

22

Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same  

DOEpatents

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

Croteau, Rodney Bruce (Pullman, WA); Burke, Charles Cullen (Moscow, ID)

2008-06-24

23

Nucleic acid arrays and methods of synthesis  

DOEpatents

The present invention generally relates to high density nucleic acid arrays and methods of synthesizing nucleic acid sequences on a solid surface. Specifically, the present invention contemplates the use of stabilized nucleic acid primer sequences immobilized on solid surfaces, and circular nucleic acid sequence templates combined with the use of isothermal rolling circle amplification to thereby increase nucleic acid sequence concentrations in a sample or on an array of nucleic acid sequences.

Sabanayagam, Chandran R. (Allston, MA); Sano, Takeshi (Needham, MA); Misasi, John (Syracuse, NY); Hatch, Anson (Seattle, WA); Cantor, Charles (Del Mar, CA)

2001-01-01

24

52906 Potassium channel nucleic acids and uses therefor  

US Patent & Trademark Office Database

The invention provides isolated nucleic acids molecules, designated 52906, 33408, or 12189 nucleic acid molecules, which encode novel potassium channel members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 52906, 33408, or 12189 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 52906, 33408, or 12189 gene has been introduced or disrupted. The invention still further provides isolated 52906, 33408, or 12189 proteins, fusion proteins, antigenic peptides and anti-52906, 33408, or 12189 antibodies. Diagnostic methods utilizing compositions of the invention are also provided.

2006-07-18

25

Replica amplification of nucleic acid arrays  

DOEpatents

A method of producing a plurality of a nucleic acid array, comprising, in order, the steps of amplifying in situ nucleic acid molecules of a first randomly-patterned, immobilized nucleic acid array comprising a heterogeneous pool of nucleic acid molecules affixed to a support, transferring at least a subset of the nucleic acid molecules produced by such amplifying to a second support, and affixing the subset so transferred to the second support to form a second randomly-patterned, immobilized nucleic acid array, wherein the nucleic acid molecules of the second array occupy positions that correspond to those of the nucleic acid molecules from which they were amplified on the first array, so that the first array serves as a template to produce a plurality, is disclosed.

Church, George M. (Brookline, MA)

2002-01-01

26

Structure of the Nucleic Acids  

Microsoft Academic Search

WE have formulated a structure for the nucleic acids which is compatible with the main features of the X-ray diagram and with the general principles of molecular structure, and which accounts satisfactorily for some of the chemical properties of the substances. The structure involves three intertwined helical polynucleotide chains. Each chain, which is formed by phosphate di-ester groups and linking

Linus Pauling; Robert B. Corey

1953-01-01

27

Applications of peptide nucleic acids  

Microsoft Academic Search

Several exciting new developments in the applications of the DNA mimic peptide nucleic acid (PNA) have been published recently. A possible breakthrough may have come in efforts to develop PNA into gene therapeutic drugs. In eukaryotic systems, antisense activity of PNAs (as peptide conjugates) has been reported in nerve cells and even in rats upon injection into the brain, and

Peter E Nielsen

1999-01-01

28

Nucleic Acids Molecular Biology Tools  

E-print Network

Biology and Genomics #12;Nucleic Acids Proteins Molecular Biology Tools Polymerase Chain Reaction (PCR The Central Dogma Key Enzymes: DNA Polymerase; RNA Polymerase; Ribosome; Reverse transcriptase Exercise of a DNA template into an RNA. Transcriptome: All mRNA in a cell Promoter In Prokaryotes: RNA polymerase

Qiu, Weigang

29

Isolation of monovalent quantum dot-nucleic acid conjugates using magnetic beads.  

PubMed

Control of the valency that is achieved in the decoration of quantum dots (QDs) remains a challenge due to the high surface area of nanoparticles. A population distribution of conjugates is formed even when reactions involve use of one-to-one molar equivalents of the ligand and QD. Monovalent conjugates are of particular interest to enable the preparation of multinanoparticle constructs that afford improved analytical functionality. Herein, a facile method for the formation and purification of QD-DNA monoconjugates (i.e., 1 DNA per QD) is described. Using diethylaminoethyl (DEAE) functionalized magnetic beads, a protocol was developed and optimized to selectively isolate QD-DNA monoconjugates from a mixture. Monoconjugates prepared with oligonucleotides as short as 19 bases and as long as 36 bases were successfully isolated. The monoconjugates were isolated in less than 5 min with isolation efficiencies between 68% and 93%, depending on the length of oligonucleotide that was used. The versatility of the method was demonstrated by purifying monoconjugates prepared from commercially available, water-soluble QDs. The isolation of monoconjugates was confirmed using agarose gel electrophoresis and single molecule fluorescence spectroscopy. Examples are provided comparing the analytical performance of monoconjugates to collections of nanoparticles of mixed valencies, indicating the significance of this separation method to prepare nanomaterials for bioassay design. PMID:24927235

Uddayasankar, Uvaraj; Zhang, Zhenfu; Shergill, Ravi T; Gradinaru, Claudiu C; Krull, Ulrich J

2014-07-16

30

Nucleic acid delivery with microbubbles and ultrasound.  

PubMed

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

Rychak, Joshua J; Klibanov, Alexander L

2014-06-01

31

Oligomeric nucleic acids as antivirals.  

PubMed

Based on the natural functions and chemical characteristics of nucleic acids, a variety of novel synthetic drugs and tools to explore biological systems have become available in recent years. To date, a great number of antisense oligonucleotides, RNA interference-based tools, CpG?containing oligonucleotides, catalytic oligonucleotides, decoys and aptamers has been produced synthetically and applied successfully for understanding and manipulating biological processes and in clinical trials to treat a variety of diseases. Their versatility and potency make them equally suited candidates for fighting viral infections. Here, we describe the different types of nucleic acid-based antivirals, their mechanism of action, their advantages and limitations, and their future prospects. PMID:21278679

Mescalchin, Alessandra; Restle, Tobias

2011-01-01

32

Nucleic acids, compositions and uses thereof  

DOEpatents

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

Preston, III, James F. (Micanopy, FL); Chow, Virginia (Gainesville, FL); Nong, Guang (Gainesville, FL); Rice, John D. (Gainesville, FL); St. John, Franz J. (Baltimore, MD)

2012-02-21

33

Self-assembling multimeric nucleic acid constructs  

DOEpatents

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

Cantor, Charles R. (Boston, MA); Niemeyer, Christof M. (Bremen, DE); Smith, Cassandra L. (Boston, MA); Sano, Takeshi (Boston, MA); Hnatowich, Donald J. (Brookline, MA); Rusckowski, Mary (Southborough, MA)

1996-01-01

34

Self-assembling multimeric nucleic acid constructs  

DOEpatents

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

Cantor, Charles R. (Boston, MA); Niemeyer, Christof M. (Bremen, DE); Smith, Cassandra L. (Boston, MA); Sano, Takeshi (Boston, MA); Hnatowich, Donald J. (Brookline, MA); Rusckowski, Mary (Southborough, MA)

1999-10-12

35

Self-assembling multimeric nucleic acid constructs  

DOEpatents

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

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

1996-10-01

36

Electronic Detection of Nucleic Acids  

PubMed Central

A novel platform for the electronic detection of nucleic acids on microarrays is introduced and shown to perform well as a selective detection system for applications in molecular diagnostics. A gold electrode in a printed circuit board is coated with a self-assembled monolayer (SAM) containing DNA capture probes. Unlabeled nucleic acid targets are immobilized on the surface of the SAM through sequence-specific hybridization with the DNA capture probe. A separate signaling probe, containing ferrocene-modified nucleotides and complementary to the target in the region adjoining the capture probe binding site, is held in close proximity to the SAM in a sandwich complex. The SAM allows electron transfer between the immobilized ferrocenes and the gold, while insulating the electrode from soluble redox species, including unbound signaling probes. Here, we demonstrate sequence-specific detection of amplicons after simple dilution of the reaction product into hybridization buffer. In addition, single nucleotide polymorphism discrimination is shown. A genotyping chip for the C282Y single nucleotide polymorphism associated with hereditary hemochromatosis is used to confirm the genotype of six patients’ DNA. In addition, a gene expression-monitoring chip is described that surveys five genes that are differentially regulated in the cellular apoptosis response. Finally, custom modification of individual electrodes through sequence-specific hybridization demonstrates the potential of this system for infectious disease diagnostics. The versatility of the electronic detection platform makes it suitable for multiple applications in diagnostics and pharmacogenetics. PMID:11333303

Umek, Robert M.; Lin, Sharon W.; Vielmetter, Jost; Terbrueggen, Robert H.; Irvine, Bruce; Yu, C. J.; Kayyem, Jon Faiz; Yowanto, Handy; Blackburn, Gary F.; Farkas, Daniel H.; Chen, Yin-Peng

2001-01-01

37

Synthesis of Glycol Nucleic Acids SynthesisofGlycolNucleicAcidsLilu Zhang, Adam E. Peritz, Patrick J. Carroll, Eric Meggers*  

E-print Network

PAPER 645 Synthesis of Glycol Nucleic Acids SynthesisofGlycolNucleicAcidsLilu Zhang, Adam E. Peritz for the automated solid phase synthesis of glycol nucleic acids (GNA) oligonucleotides and it is demonstrated of analo- gous DNA duplexes. Key words: GNA, glycol nucleic acid, glycol nucleotides, acyclic nucleic acid

Meggers, Eric

38

Molecular modeling of nucleic acid structure  

PubMed Central

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. PMID:18428873

Galindo-Murillo, Rodrigo; Bergonzo, Christina

2013-01-01

39

Conformational analysis of nucleic acids revisited: Curves+  

Microsoft Academic Search

We describe Curves+, a new nucleic acid confor- mational analysis program which is applicable to a wide range of nucleic acid structures, including those with up to four strands and with either canon- ical or modified bases and backbones. The program is algorithmically simpler and computationally much faster than the earlier Curves approach, although it still provides both helical and

R. Lavery; M. Moakher; J. H. Maddocks; D. Petkeviciute; K. Zakrzewska

2009-01-01

40

An Introduction to Peptide Nucleic Acid  

Microsoft Academic Search

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

Peter E. Nielsen; Michael Egholm

1999-01-01

41

Antisense properties of peptide nucleic acid  

Microsoft Academic Search

Peptide nucleic acid (PNA) is a nucleic acid mimic in which the deoxyribose phosphate backbone has been replaced by a pseudo-peptide polymer to which the nucleobases are linked. PNA-oligomers can be synthesized in relatively large amounts, are highly stable in biological environments, and bind complementary DNA and RNA targets with remarkably high affinity and specificity. Thus PNA possesses many of

H. Jakob Larsen; Thomas Bentin; Peter E Nielsen

1999-01-01

42

Virion nucleic acid of Ebola virus.  

PubMed Central

The virion nucleic acid of Ebola virus consists of a single-stranded RNA with a molecular weight of approximately 4.0 x 10(6). The virion RNA did not bind to oligodeoxythymidylic acid-cellulose under conditions known to bind RNAs rich in polyadenylic acid and was not infectious under conditions which yielded infectious RNA from Sindbis virus, suggesting that Ebola virus virion nucleic acid is a negative-stranded RNA. PMID:7431486

Regnery, R L; Johnson, K M; Kiley, M P

1980-01-01

43

Identification of random nucleic acid sequence aberrations using dual capture probes which hybridize to different chromosome regions  

DOEpatents

A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration. 14 figs.

Lucas, J.N.; Straume, T.; Bogen, K.T.

1998-03-24

44

Methods for analyzing nucleic acid sequences  

DOEpatents

The present invention is directed to a method of sequencing a target nucleic acid. The method provides a complex comprising a polymerase enzyme, a target nucleic acid molecule, and a primer, wherein the complex is immobilized on a support Fluorescent label is attached to a terminal phosphate group of the nucleotide or nucleotide analog. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The time duration of the signal from labeled nucleotides or nucleotide analogs that become incorporated is distinguished from freely diffusing labels by a longer retention in the observation volume for the nucleotides or nucleotide analogs that become incorporated than for the freely diffusing labels.

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

2011-05-17

45

Standardisation and reporting for nucleic acid quantification  

Microsoft Academic Search

The real-time quantitative polymerase chain reaction (qPCR) is probably the most common molecular technique in use today,\\u000a having become the method of choice for nucleic acid detection and quantification and underpinning applications ranging from\\u000a basic research through biotechnology and forensic applications to clinical diagnostics. This key technology relies on fluorescence\\u000a to detect and quantify nucleic acid amplification products, and its

Jim Huggett; Stephen A. Bustin

46

Nucleic Acid Aptamers for Living Cell Analysis  

NASA Astrophysics Data System (ADS)

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.

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

2014-06-01

47

Cell cycle nucleic acids, polypeptides and uses thereof  

DOEpatents

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

Gordon-Kamm, William J. (Urbandale, IA); Lowe, Keith S. (Johnston, IA); Larkins, Brian A. (Tucson, AZ); Dilkes, Brian R. (Tucson, AZ); Sun, Yuejin (Westfield, IN)

2007-08-14

48

Structure and Conformation of Nucleic Acids and Protein-Nucleic Acid Interactions  

E-print Network

Structure and Conformation of Nucleic Acids and Protein-Nucleic Acid Interactions Edited by M stranded RNA (ds RNA) a two-stranded antiparallel ~ structure where the 2' hydroxyl of ribose in 571 #12 the narrow groove of the ds RNA is so shallow, there is no room for a-carbons in the antiparallel ~ structure

Church, George M.

49

The simple and fast isolation of Escherichia coli O157:H7 using magnet nanoparticle embedded silica nanotube for the nucleic acid based detection.  

PubMed

In this study, we developed a simple and fast isolation tool of Escherichia coli O157:H7 (E. coli O157:H7) using a magnet nanoparticle embedded silica nanotube (MNSNT) for the detection of E. coli O157:H7 in the sample with nucleic acid based amplification. This method does not require chaotropic salt and sophisticated equipment to isolate bacteria. The E. coli O157:H7 in the sample was effectively bound to the hydrophilic surface of MNSNT in low pH binding buffer containing divalent ions and PEG without the need for expensive biological reagents such as antibodies. This E. coli O157:H7 bound MNSNT was simply isolated by a magnet, prior to adding an amplification mixture to the same micro tube without transferring the sample to another tube. Using this novel method, the detection sensitivities of E. coli O157:H7 (102 cfu/1 g of seed sprout and 102 cfu/5 mL of water) were 80% and 100%, respectively, whereas that was 0% using the commercial method. PMID:23802420

Won, Ji Yeong; Son, Sang Jun; Um, Soong Ho; Choi, Jeong-Woo; Min, Junhong

2013-05-01

50

Nucleic acid isothermal amplification technologies: a review.  

PubMed

Nucleic acid amplification technologies are used in the field of molecular biology and recombinant DNA technologies. These techniques are used as leading methods in detecting and analyzing a small quantity of nucleic acids. The polymerase chain reaction (PCR) is the most widely used method for DNA amplification for detection and identification of infectious diseases, genetic disorders and other research purposes. However, it requires a thermocycling machine to separate two DNA strands and then amplify the required fragment. Novel developments in molecular biology of DNA synthesis in vivo demonstrate the possibility of amplifying DNA in isothermal conditions without the need of a thermocycling apparatus. DNA polymerase replicates DNA with the aid of various accessory proteins. Recent identification of these proteins has enabled development of new in vitro isothermal DNA amplification methods, mimicking these in vivo mechanisms. There are several types of isothermal nucleic acid amplification methods such as transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification. In this article, we review these isothermal nucleic acid amplification technologies and their applications in molecular biological studies. PMID:18260008

Gill, Pooria; Ghaemi, Amir

2008-03-01

51

Fmoc mediated synthesis of Peptide Nucleic Acids  

Microsoft Academic Search

The syntheses of the Fmoc-protected Peptide Nucleic Acid (PNA) monomer pentafluorophenyl esters of adenine (26), cytosine (23), guanine (29) and thymine (20), and their oligomerization are described. The Fmoc PNA backbone 1 is prepared as a stable hydrochloride salt. The base acetic acids of adenine (4) and cytosine (3) were prepared by Cbz protection of the exocyclic amino groups followed

Stephen A. Thomson; John A. Josey; Rodolfo Cadilla; Micheal D. Gaul; C. Fred Hassman; Michael J. Luzzio; Adrian J. Pipe; Kathryn L. Reed; Daniel J. Ricca; Robert W. Wiethe; Stewart A. Noble

1995-01-01

52

Detection of nucleic acid sequences by invader-directed cleavage  

DOEpatents

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.

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

53

Novel biochip platform for nucleic acid analysis.  

PubMed

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. PMID:22969389

Pernagallo, Salvatore; Ventimiglia, Giorgio; Cavalluzzo, Claudia; Alessi, Enrico; Ilyine, Hugh; Bradley, Mark; Diaz-Mochon, Juan J

2012-01-01

54

The Improbability of Prebiotic Nucleic Acid Synthesis  

NASA Astrophysics Data System (ADS)

Many accounts of the origin of life assume that the spontaneous synthesis of a self-replicating nucleic acid could take place readily. Serious chemical obstacles exist, however, which make such an event extremely improbable. Prebiotic syntheses of adenine from HCN, of D, L-ribose from adenosine, and of adenosine from adenine and D-ribose have in fact been demonstrated. However these procedures use pure starting materials, afford poor yields, and are run under conditions which are not compatible with one another. Any nucleic acid components which are formed on the primitive earth would tend to hydrolyze by a number of pathways. Their polymerization would be inhibited by the presence of vast numbers of related substances which would react preferentially with them. It appears likely that nucleic acids were not formed by prebiotic routes, but are later products of evolution.

Shapiro, Robert

1984-12-01

55

Adaptive Recognition by Nucleic Acid Aptamers  

NSDL National Science Digital Library

Nucleic acid molecules play crucial roles in diverse biological processes including the storage, transport, processing, and expression of the genetic information. Nucleic acid aptamers are selected in vitro from libraries containing random sequences of up to a few hundred nucleotides. Selection is based on the ability to bind ligand molecules with high affinity and specificity. Three-dimensional structures have been determined at high resolution for a number of aptamers in complex with their cognate ligands. Structures of aptamer complexes reveal the key molecular interactions conferring specificity to the aptamer-ligand association, including the precise stacking of flat moieties, specific hydrogen bonding, and molecular shape complementarity. These basic principles of discriminatory molecular interactions in aptamer complexes parallel recognition events central to many cellular processes involving nucleic acids.

Thomas Hermann (Memorial Sloan-Kettering Cancer Center;Cellular Biochemistry and Biophysics Program); Dinshaw Patel (Memorial Sloan-Kettering Cancer Center;Cellular Biochemistry and Biophysics Program)

2000-02-04

56

Protein Nucleic Acid Interactions (Final Report, 1999-2004).  

National Technical Information Service (NTIS)

The overall goal of this collaborative project is to develop methods for analyzing protein-nucleic acid interactions. Nucleic acid-binding proteins have a central role in all aspects of genetic activity within an organism, such as transcription, replicati...

H. M. Berman, J. Thornton

2005-01-01

57

Non-instrumented nucleic acid amplification assay  

NASA Astrophysics Data System (ADS)

We have developed components of a diagnostic disposable platform that has the dual purpose of providing molecular diagnostics at the point of care (POC) as well as stabilizing specimens for further analysis via a centralized surveillance system. This diagnostic is targeted for use in low-resource settings by minimally trained health workers. The disposable device does not require any additional instrumentation and will be almost as rapid and simple to use as a lateral flow strip test - yet will offer the sensitivity and specificity of nucleic acid amplification tests (NAATs). The low-cost integrated device is composed of three functional components: (1) a sample-processing subunit that generates clean and stabilized DNA from raw samples containing nucleic acids, (2) a NA amplification subunit, and (3) visual amplicon detection sub-unit. The device integrates chemical exothermic heating, temperature stabilization using phase-change materials, and isothermal nucleic acid amplification. The aim of developing this system is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where there is no access to instrumentation. If a disease occurs, patients would be tested with the disposable in the field. A nucleic acid sample would be preserved within the spent disposable which could be sent to a central laboratory facility for further analysis if needed.

Weigl, Bernhard H.; Domingo, Gonzalo; Gerlach, Jay; Tang, Dennis; Harvey, Darrel; Talwar, Nick; Fichtenholz, Alex; van Lew, Bill; LaBarre, Paul

2008-02-01

58

Common structural features of nucleic acid polymerases  

Microsoft Academic Search

Summary Structures of multisubunit RNA polymerases strongly differ from the many known structures of single subunit DNA and RNA polymerases. However, in functional complexes of these diverse enzymes, nucleic acids take a similar course through the active center. This finding allows superposition of diverse polymerases and reveals features that are functionally equivalent. The entering DNA duplex is bent by almost

P. Cramer

2002-01-01

59

Miniaturized isothermal nucleic acid amplification, a review.  

PubMed

Micro-Total Analysis Systems (µTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented. PMID:21387067

Asiello, Peter J; Baeumner, Antje J

2011-04-21

60

Phospholipid membrane permeability of peptide nucleic acid  

Microsoft Academic Search

Phospholipid vesicles (liposomes) as membrane models have been used to study the penetration properties of peptide nucleic acid (PNA), a new DNA analog in which the nucleobases are attached to a pseudo-peptide backbone. The liposomes were characterised by carboxyfluorescein efflux, light-scattering and cryogenic transmission electron microscopy. The liposome structure was found not to be affected by the incorporation of PNA

Pernilla Wittung; Johan Kajanus; Katarina Edwards; Peter Nielsen; Bengt Nordén; Bo G. Malmström

1995-01-01

61

Cellular delivery of peptide nucleic acid (PNA)  

Microsoft Academic Search

Peptide nucleic acid (PNA) is a DNA mimic having a pseudopeptide backbone that makes it extremely stable in biological fluids. PNA binds complementary RNA and DNA with high affinity and specificity. These qualities make PNA a leading agent among ‘third generation’ antisense and antigene agents. Unfortunately, fast progress in the exploration of PNA as an experimental and therapeutical regulator of

Uffe Koppelhus; Peter E. Nielsen

2003-01-01

62

Nucleic acid amplification using modular branched primers  

Microsoft Academic Search

Methods and compositions expand the options for making primers for use in amplifying nucleic acid segments. The invention eliminates the step of custom synthesis of primers for Polymerase Chain Reactions (PCR). Instead of being custom-synthesized, a primer is replaced by a combination of several oligonucleotide modules selected from a pre-synthesized library. A modular combination of just a few oligonucleotides essentially

Levy Ulanovsky; Mugasimangalam C. Raja

2001-01-01

63

Nucleic acid-coupled colorimetric analyte detectors  

DOEpatents

The present invention relates to methods and compositions for the direct detection of analytes and membrane conformational changes through the detection of color changes in biopolymeric materials. In particular, the present invention provide for the direct colorimetric detection of analytes using nucleic acid ligands at surfaces of polydiacetylene liposomes and related molecular layer systems.

Charych, Deborah H. (Albany, CA); Jonas, Ulrich (Mainz, DE)

2001-01-01

64

Chemical Etiology of Nucleic Acid Structure  

NSDL National Science Digital Library

Systematic chemical studies indicate that the capability of Watson-Crick base-pairing is widespread among potentially natural nucleic acid alternatives taken from RNA's close structural neighborhood. A comparison of RNA and such alternatives with regard to chemical properties that are fundamental to the biological function of RNA provides chemical facts that may contain clues to RNA's origin.

Albert Eschenmoser (The Scripps Research Institute ;)

1999-06-25

65

Antigene, Ribozyme and Aptamer Nucleic Acid Drugs: Progress and Prospects  

Microsoft Academic Search

Nucleic acids are increasingly being considered for therapeutic uses, either to interfere with the function of specific nucleic acids or to bind specific proteins. Three types of nucleic acid drugs are discussed in this review: aptamers, compounds which bind specific proteins; triplex forming (antigene) compounds; which bind double stranded DNA; and ribozymes (catalytic RNA), which bind and cleave RNA targets.

Robert A. Stull

1995-01-01

66

AminoglycosideNucleic Acid Interactions: The Case for Neomycin  

E-print Network

Aminoglycoside­Nucleic Acid Interactions: The Case for Neomycin Dev P.Arya ( ) 461 Hunter Aminoglycosides and Nucleic Acids: The Attraction for RNA? . . . . . . . . . 152 2.1 The Need for New Approaches: DNA vs RNA Recognition . . . . . . . . . . . 152 3 The Nucleic Acid Triplex: Role of Aminoglycosides

Stuart, Steven J.

67

Synthesis of Neomycin-DNA/ Peptide Nucleic Acid  

E-print Network

Synthesis of Neomycin-DNA/ Peptide Nucleic Acid Conjugates I. Charles and Dev P. Arya Laboratory conjugates of DNA and peptide nucleic acid (PNA) is reported. The DNA and PNA conjugates were prepared deterrent in forming duplexes and triplexes, peptide nucleic acid (PNA), a neutral backbone containing

Stuart, Steven J.

68

Nucleic acid analysis using terminal-phosphate-labeled nucleotides  

DOEpatents

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

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

2008-04-22

69

Conformational Flexibility of Pyrimidine Ring in Nucleic Acid Bases  

Microsoft Academic Search

\\u000a Nucleic acid bases (NABs) have been considered for many years to be planar and conformationally rigid. However, recently,\\u000a two possible sources of nucleobases nonplanarity have been found. Ab initio quantum-chemical calculations using large basis\\u000a sets augmented by inclusion of electron correlation and recent experimental studies revealed that amino groups in isolated\\u000a cytosine, guanine, and adenine adopt a nonplanar trigonal-pyramidal configuration.

Oleg V. Shishkin; Leonid Gorb; Jerzy Leszczynski

2010-01-01

70

Diagnostic applications of nucleic acid circuits.  

PubMed

CONSPECTUS: While the field of DNA computing and molecular programming was engendered in large measure as a curiosity-driven exercise, it has taken on increasing importance for analytical applications. This is in large measure because of the modularity of DNA circuitry, which can serve as a programmable intermediate between inputs and outputs. These qualities may make nucleic acid circuits useful for making decisions relevant to diagnostic applications. This is especially true given that nucleic acid circuits can potentially directly interact with and be triggered by diagnostic nucleic acids and other analytes. Chemists are, by and large, unaware of many of these advances, and this Account provides a means of touching on what might seem to be an arcane field. We begin by explaining nucleic acid amplification reactions that can lead to signal amplification, such as catalytic hairpin assembly (CHA) and the hybridization chain reaction (HCR). In these circuits, a single-stranded input acts on kinetically trapped substrates via exposed toeholds and strand exchange reactions, refolding the substrates and allowing them to interact with one another. As multiple duplexes (CHA) or concatemers of increasing length (HCR) are generated, there are opportunities to couple these outputs to different analytical modalities, including transduction to fluorescent, electrochemical, and colorimetric signals. Because both amplification and transduction are at their root dependent on the programmability of Waston-Crick base pairing, nucleic acid circuits can be much more readily tuned and adapted to new applications than can many other biomolecular amplifiers. As an example, robust methods for real-time monitoring of isothermal amplification reactions have been developed recently. Beyond amplification, nucleic acid circuits can include logic gates and thresholding components that allow them to be used for analysis and decision making. Scalable and complex DNA circuits (seesaw gates) capable of carrying out operations such as taking square roots or implementing neural networks capable of learning have now been constructed. Into the future, we can expect that molecular circuitry will be designed to make decisions on the fly that reconfigure diagnostic devices or lead to new treatment options. PMID:24828239

Jung, Cheulhee; Ellington, Andrew D

2014-06-17

71

Easy to use and rapid isolation and detection of a viral nucleic acid by using paramagnetic microparticles and carbon nanotubes-based screen-printed electrodes  

Microsoft Academic Search

A method that is easy to use, rapid, with a low cost of detecting viral nucleic acid in a biological sample represents the\\u000a essential tool in targeted therapy. In this study, we report the use of paramagnetic microparticles covered by streptavidin\\u000a and modified by an oligonucleotide probe with a specific viral sequence labeled by biotin to detect human immunodeficiency\\u000a virus

Vojtech Adam; Dalibor Huska; Jaromir Hubalek; Rene Kizek

2010-01-01

72

Development of a rapid total nucleic acid extraction method for the isolation of hepatitis A virus from fresh produce.  

PubMed

Recently, there have been increasing reports of foodborne illnesses associated with the consumption of fresh produce. Among these, hepatitis A virus (HAV) remains epidemiologically important and has been continually implicated in several outbreaks. We describe a rapid method (<8h) for the isolation and subsequent detection with real-time quantitative PCR (RT-qPCR) of the HAV HM-175 cytopathic strain seeded onto baby spinach and sliced tomatoes using a total RNA extraction method, utilizing a high concentration (4M) guanidine thiocyanate buffer. Consistent detection of HAV genome from both produce items was achieved at an inoculation level of 3×10³ PFU/25 g of food, with less consistent detection achieved at 3×10² PFU/25g. Initial studies revealed that a final precipitation of recovered RNA with potassium acetate to reduce carryover of polysaccharides and the addition of polyvinylpyrrolidone to remove polyphenolics in spinach were essential. For tomatoes, virus isolation was achieved with the incorporation of either an elution step with a high pH Tris-glycine-beef extract (TGBE) buffer or with an enzymatic digestion with pectinase. We also describe the development of a protocol for the detection of HAV from tomatoes utilizing a Luminex® microbead-based suspension array. The results correlated well with the RT-qPCR assay suggesting the feasibility of the Bioplex® as a detection platform for viruses isolated from foods. PMID:23334093

Hida, Kaoru; Kulka, Michael; Papafragkou, Efstathia

2013-02-15

73

Conformational analysis of nucleic acids revisited: Curves+.  

PubMed

We describe Curves+, a new nucleic acid conformational analysis program which is applicable to a wide range of nucleic acid structures, including those with up to four strands and with either canonical or modified bases and backbones. The program is algorithmically simpler and computationally much faster than the earlier Curves approach, although it still provides both helical and backbone parameters, including a curvilinear axis and parameters relating the position of the bases to this axis. It additionally provides a full analysis of groove widths and depths. Curves+ can also be used to analyse molecular dynamics trajectories. With the help of the accompanying program Canal, it is possible to produce a variety of graphical output including parameter variations along a given structure and time series or histograms of parameter variations during dynamics. PMID:19625494

Lavery, R; Moakher, M; Maddocks, J H; Petkeviciute, D; Zakrzewska, K

2009-09-01

74

Conformational analysis of nucleic acids revisited: Curves+  

PubMed Central

We describe Curves+, a new nucleic acid conformational analysis program which is applicable to a wide range of nucleic acid structures, including those with up to four strands and with either canonical or modified bases and backbones. The program is algorithmically simpler and computationally much faster than the earlier Curves approach, although it still provides both helical and backbone parameters, including a curvilinear axis and parameters relating the position of the bases to this axis. It additionally provides a full analysis of groove widths and depths. Curves+ can also be used to analyse molecular dynamics trajectories. With the help of the accompanying program Canal, it is possible to produce a variety of graphical output including parameter variations along a given structure and time series or histograms of parameter variations during dynamics. PMID:19625494

Lavery, R.; Moakher, M.; Maddocks, J. H.; Petkeviciute, D.; Zakrzewska, K.

2009-01-01

75

Nucleic Acid and the Beginning of Meiosis  

Microsoft Academic Search

A NEW acetic-lacmoid squash method of preparation followed by Feulgen staining of the desoxyribose nucleic acid1 has made it possible to trace certain chemical and physical changes undergone by the nucleus during the critical stages at the beginning of meiosis. The most favourable material is found in some liliaceous plants with large nuclei. Both types of mother-cell are shown up

C. D. Darlington; L. F. La Cour

1946-01-01

76

Peptide nucleic acid delivery to human mitochondria  

Microsoft Academic Search

Peptide nucleic acids (PNAs) are synthetic polynucleobase molecules, which bind to DNA and RNA with high affinity and specificity. Although PNAs have enormous potential as anti-sense agents, the success of PNA-mediated gene therapy will require efficient cellular uptake and sub-cellular trafficking. At present these mechanisms are poorly understood. To address this, we have studied the uptake of biotinylated PNAs into

P F Chinnery; R W Taylor; K Diekert; R Lill; D M Turnbull; R N Lightowlers

1999-01-01

77

Therapeutic Potential of Antisense Nucleic Acid Molecules  

NSDL National Science Digital Library

Elucidation of many disease-related signal transduction and gene expression pathways has provided unparalleled opportunities for the development of targeted therapeutics. The types of molecules in development are increasingly varied and include small-molecule enzyme inhibitors, humanized antibodies to cell surface receptors, and antisense nucleic acids for silencing the expression of specific genes. This Perspective reviews the basis for various antisense strategies for modulating gene expression, including RNA interference, and discusses the prospects for their clinical use.

Joanna B. Opalinska (Pommeranian Medical Academy;Department of Hematology REV); Alan M. Gewirtz (University of Pennsylvania School of Medicine;Division of Hematology and Oncology REV)

2003-10-28

78

Carbohydrate Polymers for Nonviral Nucleic Acid Delivery  

PubMed Central

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

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

2014-01-01

79

Optimizing the specificity of nucleic acid hybridization  

PubMed Central

The specific hybridization of complementary sequences is an essential property of nucleic acids, enabling diverse biological and biotechnological reactions and functions. However, the specificity of nucleic acid hybridization is compromised for long strands, except near the melting temperature. Here, we analytically derived the thermodynamic properties of a hybridization probe that would enable near-optimal single-base discrimination and perform robustly across diverse temperature, salt and concentration conditions. We rationally designed ‘toehold exchange’ probes that approximate these properties, and comprehensively tested them against five different DNA targets and 55 spurious analogues with energetically representative single-base changes (replacements, deletions and insertions). These probes produced discrimination factors between 3 and 100+ (median, 26). Without retuning, our probes function robustly from 10 °C to 37 °C, from 1 mM Mg2+ to 47 mM Mg2+, and with nucleic acid concentrations from 1 nM to 5 ?M. Experiments with RNA also showed effective single-base change discrimination. PMID:22354435

Zhang, David Yu; Chen, Sherry Xi; Yin, Peng

2014-01-01

80

[Circulating nucleic acids and in vitro fertilization].  

PubMed

During the last years, the use of circulating nucleic acids (microRNAs and cell-free DNA) as diagnostic and/or prognostic tools in cancerology was widely documented. Likewise, in obstetrics and gynecology, the development of non-invasive prenatal testing based on the assessment of these biomarkers confirmed their growing interest in this speciality. In human reproduction, several studies were interested in the microRNAs, small non-coding RNA sequences, present in the ovarian follicle and their implication in folliculogenesis. Some of these microRNAs, as well as the vesicles which transport them, are easily detectable in the bloodstream and could be used as reliable biomarkers of interest in infertility care. Cell-free DNA level varies according to physiopathology and reflect the proportion of apoptotic and/or necrotic events occurring in the body. As a result, its quantification could give an additional help to the practitioners for ovarian functional status evaluation. Furthermore, these circulating nucleic acids could also constitute new predictive biomarkers of oocyte and/or embryo quality and represent a promising perspective for the prevention of in vitro fertilization implantation failures. In conclusion, these circulating nucleic acids open the way to the development of new diagnostic and/or prognostic innovative tests in order to improve in vitro fertilization outcomes. PMID:25155829

Scalici, E; Traver, S; Mullet, T; Ferrières, A; Monforte, M; Vintejoux, E; Hamamah, S

2014-10-01

81

Determination of HER-2/neu amplification and expression in tumor tissue and cultured cells using a simple, phenol free method for nucleic acid isolation.  

PubMed

A rapid, simple and non-toxic procedure for the simultaneous isolation of DNA and RNA from tumor tissue and cells grown in vitro is described. Guanidinium isothiocyanate was used for homogenization of tumor tissue and for cell lysis. Separation of proteins, DNA and RNA was carried out by isopycnic centrifugation in cesium trifluoroacetate. DNA was further purified by salting out residual protein. Nucleic acids prepared by this method from 47 primary human carcinomas and 17 human cell lines were analysed for amplification and expression of the HER-2/neu proto-oncogene. 2- to 10-fold amplification of HER-2/neu was noted in 7/22 mammary carcinomas (32%) and in 4/14 ovarian carcinomas (28%). No amplification of the proto-oncogene was found in 4 laryngeal carcinomas, 1 pharyngeal carcinoma, 2 retrolingual carcinomas, 3 gastric carcinomas and 1 kidney carcinoma. HER-2/neu overexpression was observed in 6/22 of mammary carcinomas (27%) and 7/14 of ovarian carcinomas (50%). No overexpression was found in all other carcinomas studied. Concordance between amplification and overexpression was noted in 3 mammary and 4 ovarian carcinomas, respectively. 3 mammary and 3 ovarian carcinomas showed overexpression without amplification. 5 human mammary carcinoma cell lines showed both amplification and overexpression of HER-2/neu. In two mammary carcinoma cell lines (MDA MB-453 and ZR 75-1) overexpression was noted without amplification of the proto-oncogene. These data combine to suggest that mechanisms other than gene amplification may also lead to overexpression of the HER-2/neu protooncogene in cancer cells. PMID:1699198

Kury, F D; Schneeberger, C; Sliutz, G; Kubista, E; Salzer, H; Medl, M; Leodolter, S; Swoboda, H; Zeillinger, R; Spona, J

1990-09-01

82

Helicase-dependent amplification of nucleic acids.  

PubMed

Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. PMID:24510297

Cao, Yun; Kim, Hyun-Jin; Li, Ying; Kong, Huimin; Lemieux, Bertrand

2013-01-01

83

The Nucleic Acid Database: new features and capabilities.  

PubMed

The Nucleic Acid Database (NDB) (http://ndbserver.rutgers.edu) is a web portal providing access to information about 3D nucleic acid structures and their complexes. In addition to primary data, the NDB contains derived geometric data, classifications of structures and motifs, standards for describing nucleic acid features, as well as tools and software for the analysis of nucleic acids. A variety of search capabilities are available, as are many different types of reports. This article describes the recent redesign of the NDB Web site with special emphasis on new RNA-derived data and annotations and their implementation and integration into the search capabilities. PMID:24185695

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

2014-01-01

84

CRC handbook of chromatography: Nucleic acids and related compounds  

SciTech Connect

This book's contents include: Structure Elucidation of Nucleic Acid Components; Fundamentals of HPLC; Analysis of Nucleic Acids and Oligonucleotides; Extraction of Nucleic Acids from Tissues; Gel Filtration Chromatography of RNAs and DNS Fragments; Separation of tRNAs and Oligonucleotides by Mixed Mode Chromatography; Anion-Exchange and Reversed-Phase HPLC of Synthetic Oligonucleotides; Nucleic Acid Components in Biological Fluids; RPLC Separation of RNA and DNA Hydrolysates; Nucleotides in Tissue Extracts; and Determination of Adenine Nucleotides and Creatine Phosphate in Various Mammalian Tissues.

Krstulovic, A.M.

1987-01-01

85

Nucleic acid amplification using modular branched primers  

DOEpatents

Methods and compositions expand the options for making primers for use in amplifying nucleic acid segments. The invention eliminates the step of custom synthesis of primers for Polymerase Chain Reactions (PCR). Instead of being custom-synthesized, a primer is replaced by a combination of several oligonucleotide modules selected from a pre-synthesized library. A modular combination of just a few oligonucleotides essentially mimics the performance of a conventional, custom-made primer by matching the sequence of the priming site in the template. Each oligonucleotide module has a segment that matches one of the stretches within the priming site.

Ulanovsky, Levy (Westmont, IL)

2001-01-01

86

Nucleic acid probes in diagnostic medicine  

NASA Technical Reports Server (NTRS)

The need for improved diagnostic procedures is outlined and variations in probe technology are briefly reviewed. A discussion of the application of probe technology to the diagnosis of disease in animals and humans is presented. A comparison of probe versus nonprobe diagnostics and isotopic versus nonisotopic probes is made and the current state of sequence amplification is described. The current market status of nucleic acid probes is reviewed with respect to their diagnostic application in human and veterinary medicine. Representative product examples are described and information on probes being developed that offer promise as future products is discussed.

Oberry, Phillip A.

1991-01-01

87

ORIGIN OF LIFE: A Simpler Nucleic Acid  

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. What was the genetic material of the earliest life forms on Earth if it was not RNA? As Orgel explains in his Perspective, the answer may be simpler nucleic acid polymers perhaps like the RNA analogs called (L)-a-threofuranosyl oligonucleotides or TNAs (Schöning et al.). These molecules have threose rather than ribose in their sugar-phosphate backbones and yet retain many of the properties of RNA including the ability to pair up in double helices.

Leslie Orgel (Salk Institute for Biomedical Study;)

2000-11-17

88

Nucleic acid interactions with pyrite surfaces  

NASA Astrophysics Data System (ADS)

The study of the interaction of nucleic acid molecules with mineral surfaces is a field of growing interest in organic chemistry, origin of life, material science and biotechnology. We have characterized the adsorption of single-stranded peptide nucleic acid (ssPNA) on a natural pyrite surface, as well as the further adsorption of ssDNA on a PNA-modified pyrite surface. The characterization has been performed by means of reflection absorption infrared spectroscopy (RAIRS), atomic force microscopy (AFM) and X-ray photoemission spectroscopy (XPS) techniques. The N(1s) and S(2p) XPS core level peaks of PNA and PNA + DNA have been decomposed in curve-components that we have assigned to different chemical species. RAIRS spectra recorded for different concentrations show the presence of positive and negative adsorption bands, related to the semiconducting nature of the surface. The combination of the information gathered by these techniques confirms that PNA adsorbs on pyrite surface, interacting through nitrogen-containing groups of the nucleobases and the iron atoms of the surface, instead of the thiol group of the molecule. The strong PNA/pyrite interaction inhibits further hybridization of PNA with complementary ssDNA, contrary to the behavior reported on gold surfaces.

Mateo-Martí, E.; Briones, C.; Rogero, C.; Gomez-Navarro, C.; Methivier, Ch.; Pradier, C. M.; Martín-Gago, J. A.

2008-09-01

89

Affinity sorbents containing nucleic acids and their fragments  

NASA Astrophysics Data System (ADS)

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.

Shishkina, I. G.; Levina, A. S.; Zarytova, V. F.

2001-06-01

90

Charge Transfer through Single-Stranded Peptide Nucleic Acid Composed of Thymine Nucleotides  

E-print Network

Charge Transfer through Single-Stranded Peptide Nucleic Acid Composed of Thymine Nucleotides Amit; In Final Form: February 18, 2008 Self-assembled monolayers (SAMs) of single-stranded peptide nucleic acids. Peptide nucleic acid (PNA) is an analo

Borguet, Eric

91

XVIth Symposium on Chemistry of Nucleic Acid Components.  

PubMed

SCNAC, the XVIth Symposium on Chemistry of Nucleic Acid Components, was held in ?eský Krumlov (Czech Republic) in June. This year's symposium, which covered the chemistry, biochemistry, biophysics and chemical biology of nucleobases, nucleosides, nucleotides and nucleic acids attracted more than 150 participants from 21 countries to its lectures, oral communications and poster presentations. PMID:25250893

Gheerardijn, Vicky; Madder, Annemieke

2014-11-24

92

Nucleic acid and protein mass mapping by live-cell  

E-print Network

structure. Finally, because of the shorter wavelength illumination, the theoretical spatial resolutionNucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy Benjamin J Zeskind1 the intensity of each pixel into an estimate of mass, deep-UV microscopy images generate maps of nucleic acid

Cai, Long

93

Probing Ribonuclease A Using Non-Natural Nucleic Acids  

E-print Network

Probing Ribonuclease A Catalysis Using Non-Natural Nucleic Acids By Bradley Roger Kelemen Ribonuclease A Catalysis Using Non-Natural Nucleic Acids submitted to the Graduate School of the University of Graduate School #12;A~t{NOWLEDGMENTS The Raines lab graduate students, undergraduate students

Raines, Ronald T.

94

Coupling Translocation with Nucleic Acid Unwinding by NS3 Helicase  

E-print Network

Coupling Translocation with Nucleic Acid Unwinding by NS3 Helicase Jin Yu1 , Wei Cheng2 , Carlos to be on the order of 10 s-1 . The generic features of coupling single-stranded nucleic acid translocation metabolism.1­3 Like most motor proteins, helicases use the free energy from NTP hydrolysis to translocate

Oster, George

95

Nucleic acid based fluorescent sensor for mercury detection  

DOEpatents

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.

Lu, Yi; Liu, Juewen

2013-02-05

96

Structural and biological properties of peptide nucleic acid (PNA)  

Microsoft Academic Search

The chemical, physical and biological properties of PNA (peptide nucleic acid) is briefly reviewed. In particular, recent X-ray crystallography and NMR structural data on PNA complexes are discussed. Furthermore, effects of backbone or nucleobase modifications on the PNA nucleic acid hybridization properties are discussed.

Peter E. Nielsen

1998-01-01

97

Peptide Nucleic Acid-Assisted Topological Labeling of Duplex DNA  

Microsoft Academic Search

Peptide nucleic acids (PNAs) are a family of synthetic polyamide mimics of nucleic acids that offer a variety of applications. Pyrimidine bis-PNAs can be used for rational design of novel interlocked DNA nanostructures, earring labels, representing locked pseudorotaxanes or locked catenanes. These structures are created through DNA ligase-mediated catenation of duplex DNA with a circularized oligonucleotide tag at a designated

Vadim V. Demidov; Heiko Kuhn; Irina V. Lavrentieva-Smolina; Maxim D. Frank-Kamenetskii

2001-01-01

98

Purification of nucleic acids using isotachophoresis.  

PubMed

Reviewed are methods of nucleic acid (NA) extraction and sample preparation using an electrophoretic purification and focusing method called isotachophoresis (ITP). ITP requires no special surface chemistries or geometric structures, and can be achieved in a compact system with no moving parts. ITP is also compatible with a wide range of samples and lysing methods. Described are general principles of ITP, considerations around the application of ITP to biological samples (e.g., blood, urine and saliva), ITP electrolyte design considerations for fast and selective NA purification, and examples of ITP compatible lysing methods. Several of the challenges associated with purification of NAs are presented as well as methods to address these. Lastly, specific examples of lysing methods and ITP chemistries are described for purification of NA including host and pathogenic DNA, pathogenic rRNA, and host micro-RNA from complex sample matrices. PMID:24444800

Rogacs, Anita; Marshall, Lewis A; Santiago, Juan G

2014-03-28

99

Macromolecular Structure Description: This course covers the principles of protein and nucleic acid structure, stability  

E-print Network

of Biopolymers Amino Acids The Peptide Bond Protein Rotamers: Ramachandran plots The Nucleic Acid Bases and nucleic acid structure, stability and dynamics. Topics will include interactions, conformations, forces The Nucleic Acid Backbone Nucleic Acid Rotamers Introduction to PYMOL: visualization software Introduction

Sherrill, David

100

Methods and compositions for efficient nucleic acid sequencing  

DOEpatents

Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

Drmanac, Radoje (850 E. Greenwich Pl., Palo Alto, CA 94303)

2002-01-01

101

Lateral flow biosensors for the detection of nucleic acid.  

PubMed

The detection of nucleic acid is of central importance for the diagnosis of genetic diseases, infectious agents, and biowarfare agents. Traditional strategies and technologies for nucleic acid detection are time-consuming and labor-intensive. Recently, isothermal strand-displacement reaction-based lateral flow biosensors have attracted a great deal of research interest because they are sensitive, simple, fast, and easy to use. Here, we describe a lateral flow biosensor based on isothermal strand-displacement polymerase reaction and gold nanoparticles for the visual detection of nucleic acid. PMID:24026695

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

2013-01-01

102

Immune sensing of nucleic acids in inflammatory skin diseases.  

PubMed

Endosomal and cytosolic nucleic acid receptors are important immune sensors required for the detection of infecting or replicating viruses. The intracellular location of these receptors allows viral recognition and, at the same time, avoids unnecessary immune activation to self-nucleic acids that are continuously released by dying host cells. Recent evidence, however, indicates that endogenous factors such as anti-microbial peptides have the ability to break this protective mechanism. Here, we discuss these factors and illustrate how they drive inflammatory responses by promoting immune recognition of self-nucleic acids in skin wounds and inflammatory skin diseases such as psoriasis and lupus. PMID:25224103

Demaria, Olivier; Di Domizio, Jeremy; Gilliet, Michel

2014-09-01

103

Entrapment of nucleic acids in liposomes.  

PubMed

The entrapment efficiency of three main methods used in the literature for the encapsulation of nucleic acids in liposomes were studied using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. In particular the reverse phase method, the dehydration/rehydration method, and the freeze/thawing method were compared to each other under standardised conditions, i.e. using in every case the same concentration of guest molecules (DNA, tRNA and ATP as low molecular weight analogue) and equally extruded liposomes. The percentage of entrapment strictly referred to the material localized inside the liposomes, i.e. particular care was devoted to ruling out the contribution of the nucleic acid material bound to the outer surface of the liposomes: this was eliminated by extensive enzymatic digestion prior to column chromatography. Depending on the conditions used, the percentage of the entrapped material varied between 10 and 54% of the initial amount. Further, the encapsulation efficiency was markedly affected by the salt concentration, by the size of liposomes, but to a lower degree by the molecular weight of the guest molecules. In general, we observed that the freeze/thawing encapsulation procedure was the most efficient one. In a second part of the work the freeze/thawing method was applied to encapsulate DNA (369 bp and 3368 bp, respectively) using liposomes obtained from POPC mixed with 1-10% charged cosurfactant, i.e. phosphatidylserine (PS) or didodecyldimethylammonium bromide (DDAB), respectively. Whereas PS had no significant effect, the entrapment efficiency went up to 60% in POPC/DDAB (97.5:2.5) liposomes. The large entrapment efficiency of DNA permits spectroscopic investigations of the DNA encapsulated in the water pool of the liposomes. UV absorption and circular dichroism spectra were practically the same as in water, indicating no appreciable perturbation of the electronic transitions or of the conformation of the entrapped biopolymer. This was in contrast to the DNA bound externally to the POPC/DDAB liposomes which showed significant spectral changes with respect to DNA dissolved in water. PMID:9370243

Monnard, P A; Oberholzer, T; Luisi, P

1997-10-01

104

Efficient and Sequence-Specific DNA-Templated Polymerization of Peptide Nucleic Acid Aldehydes  

E-print Network

Efficient and Sequence-Specific DNA-Templated Polymerization of Peptide Nucleic Acid Aldehydes. Peptide nucleic acids (PNAs) are attractive candidates for synthetic polymer evolution because and sequence-specific nucleic acid-templated polymerization of proteins and nucleic acids is a fundamental

Liu, David R.

105

Molecular Modeling of Nucleic Acid Structure: Energy and Sampling  

PubMed Central

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. PMID:18428876

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

2013-01-01

106

Selenium Derivatization of Nucleic Acids for Crystallography  

SciTech Connect

The high-resolution structure of the DNA (5'-GTGTACA-C-3') with the selenium derivatization at the 2'-position of T2 was determined via MAD and SAD phasing. The selenium-derivatized structure (1.28 {angstrom} resolution) with the 2'-Se modification in the minor groove is isomorphorous to the native structure (2.0 {angstrom}). To directly compare with the conventional bromine derivatization, we incorporated bromine into the 5-postion of T4, determined the bromine-derivatized DNA structure at 1.5 {angstrom} resolution, and found that the local backbone torsion angles and solvent hydration patterns were altered in the structure with the Br incorporation in the major groove. Furthermore, while the native and Br-derivatized DNAs needed over a week to form reasonable-size crystals, we observed that the Se-derivatized DNAs grew crystals overnight with high-diffraction quality, suggesting that the Se derivatization facilitated the crystal formation. In addition, the Se-derivatized DNA sequences crystallized under a broader range of buffer conditions, and generally had a faster crystal growth rate. Our experimental results indicate that the selenium derivatization of DNAs may facilitate the determination of nucleic acid X-ray crystal structures in phasing and high-quality crystal growth. In addition, our results suggest that the Se derivatization can be an alternative to the conventional Br derivatization.

Jiang,J.; Sheng, J.; Carrasco, N.; Huang, Z.

2007-01-01

107

A self-replicating peptide nucleic acid.  

PubMed

While the non-enzymatic ligation and template-directed synthesis of peptide nucleic acids (PNA) have been reported since 1995, a case of self-replication of PNA has not been achieved yet. Here, we present evidence for autocatalytic feedback in a template directed synthesis of a self-complementary hexa-PNA from two trimeric building blocks. The course of the reaction was monitored in the presence of increasing initial concentrations of the product by RP-HPLC. Kinetic modeling with the SimFit program revealed parabolic growth characteristics. The observed template effect, as well as the rate of ligation, was significantly influenced by nucleophilic catalysts, pH value, and uncharged co-solvents. Systematic optimization of the reaction conditions allowed us to increase the autocatalytic efficiency of the system by two orders of magnitude. Our findings contribute to the hypothesis that PNA may have served as a primordial genetic molecule and was involved in a potential precursor of a RNA world. PMID:25065957

Plöger, Tobias A; von Kiedrowski, Günter

2014-09-21

108

Selenium derivatization of nucleic acids for crystallography  

PubMed Central

The high-resolution structure of the DNA (5?-GTGTACA-C-3?) with the selenium derivatization at the 2?-position of T2 was determined via MAD and SAD phasing. The selenium-derivatized structure (1.28 Å resolution) with the 2?-Se modification in the minor groove is isomorphorous to the native structure (2.0 Å). To directly compare with the conventional bromine derivatization, we incorporated bromine into the 5-postion of T4, determined the bromine-derivatized DNA structure at 1.5 Å resolution, and found that the local backbone torsion angles and solvent hydration patterns were altered in the structure with the Br incorporation in the major groove. Furthermore, while the native and Br-derivatized DNAs needed over a week to form reasonable-size crystals, we observed that the Se-derivatized DNAs grew crystals overnight with high-diffraction quality, suggesting that the Se derivatization facilitated the crystal formation. In addition, the Se-derivatized DNA sequences crystallized under a broader range of buffer conditions, and generally had a faster crystal growth rate. Our experimental results indicate that the selenium derivatization of DNAs may facilitate the determination of nucleic acid X-ray crystal structures in phasing and high-quality crystal growth. In addition, our results suggest that the Se derivatization can be an alternative to the conventional Br derivatization. PMID:17169989

Jiang, Jiansheng; Sheng, Jia; Carrasco, Nicolas; Huang, Zhen

2007-01-01

109

Optimization of Encoded Hydrogel Particles for Nucleic Acid Quantification  

E-print Network

The accurate quantification of nucleic acids is of utmost importance for clinical diagnostics, drug discovery, and basic science research. These applications require the concurrent measurement of multiple targets while ...

Pregibon, Daniel C.

110

Dioxaphosphorinane-Constrained Nucleic Acid Dinucleotides as Tools for Structural Tuning of Nucleic Acids  

PubMed Central

We describe a rational approach devoted to modulate the sugar-phosphate backbone geometry of nucleic acids. Constraints were generated by connecting one oxygen of the phosphate group to a carbon of the sugar moiety. The so-called dioxaphosphorinane rings were introduced at key positions along the sugar-phosphate backbone allowing the control of the six-torsion angles ? to ? defining the polymer structure. The syntheses of all the members of the D-CNA family are described, and we emphasize the effect on secondary structure stabilization of a couple of diastereoisomers of ?,?-D-CNA exhibiting wether B-type canonical values or not. PMID:23150809

Catana, Dan-Andrei; Renard, Brice-Loic; Maturano, Marie; Payrastre, Corinne; Tarrat, Nathalie; Escudier, Jean-Marc

2012-01-01

111

Molecular Modeling of Nucleic Acid Structure: Setup and Analysis  

PubMed Central

The last in a set of units by these authors, this unit addresses some important remaining questions about molecular modeling of nucleic acids. It describes how to choose an appropriate molecular mechanics force field; how to set up and equilibrate the system for accurate simulation of a nucleic acid in an explicit solvent by molecular dynamics or Monte Carlo simulation; and provides some information about how to analyze molecular dynamics trajectories. PMID:18428869

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

2014-01-01

112

Encapsulation of Nucleic Acids and Opportunities for Cancer Treatment  

Microsoft Academic Search

The development of nucleic acid drugs for the treatment of various cancers has shown great promise in recent years. However,\\u000a efficient delivery of these drugs to target cells remains a significant challenge towards the successful development of such\\u000a therapies. This review provides a comprehensive overview of encapsulation technologies being developed for the delivery of\\u000a nucleic acid-based anti-cancer agents. Both micro

Lisa Brannon-Peppas; Bilal Ghosn; Krishnendu Roy; Kenneth Cornetta

2007-01-01

113

Cigarette smoke induces nucleic-acid oxidation in lung fibroblasts.  

PubMed

Oxidative stress is widely proposed as a pathogenic mechanism for chronic obstructive pulmonary disease (COPD), but the molecular pathway connecting oxidative damage to tissue destruction remains to be fully defined. We suggest that reactive oxygen species (ROS) oxidatively damage nucleic acids, and this effect requires multiple repair mechanisms, particularly base excision pathway components 8-oxoguanine-DNA glycosylase (OGG1), endonuclease III homologue 1 (NTH1), and single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), as well as the nucleic acid-binding protein, Y-box binding protein 1 (YB1). This study was therefore designed to define the levels of nucleic-acid oxidation and expression of genes involved in the repair of COPD and in corresponding models of this disease. We found significant oxidation of nucleic acids localized to alveolar lung fibroblasts, increased levels of OGG1 mRNA expression, and decreased concentrations of NTH1, SMUG1, and YB1 mRNA in lung samples from subjects with very severe COPD compared with little or no COPD. Mice exposed to cigarette smoke exhibited a time-dependent accumulation of nucleic-acid oxidation in alveolar fibroblasts, which was associated with an increase in OGG1 and YB1 mRNA concentrations. Similarly, human lung fibroblasts exposed to cigarette smoke extract exhibited ROS-dependent nucleic-acid oxidation. The short interfering RNA (siRNA)-dependent knockdown of OGG1 and YB1 expression increased nucleic-acid oxidation at the basal state and after exposure to cigarette smoke. Together, our results demonstrate ROS-dependent, cigarette smoke-induced nucleic-acid oxidation in alveolar fibroblasts, which may play a role in the pathogenesis of emphysema. PMID:20008282

Deslee, Gaetan; Adair-Kirk, Tracy L; Betsuyaku, Tomoko; Woods, Jason C; Moore, Carla H; Gierada, David S; Conradi, Susan H; Atkinson, Jeffrey J; Toennies, Holly M; Battaile, John T; Kobayashi, Dale K; Patterson, G Alexander; Holtzman, Michael J; Pierce, Richard A

2010-11-01

114

Nucleic Acid Transport in Plant-Pathogen Interactions  

Microsoft Academic Search

\\u000a Transport of nucleic acid molecules is central to many plant-pathogen interactions. Nucleic acids are transported between\\u000a cells when plant viruses move their genomes from the infected into adjacent uninfected cells through plant intercellular connections,\\u000a the plasmodesmata. DNA and RNA molecules are also transported from the host cell cytoplasm into the nucleus during many viral\\u000a infections. In addition, nuclear import of

Robert Lartey; Vitaly Citovsky

115

Single-stranded nucleic acids promote SAMHD1 complex formation.  

PubMed

SAM domain and HD domain-containing protein 1 (SAMHD1) is a dGTP-dependent triphosphohydrolase that degrades deoxyribonucleoside triphosphates (dNTPs) thereby limiting the intracellular dNTP pool. Mutations in SAMHD1 cause Aicardi-Goutières syndrome (AGS), an inflammatory encephalopathy that mimics congenital viral infection and that phenotypically overlaps with the autoimmune disease systemic lupus erythematosus. Both disorders are characterized by activation of the antiviral cytokine interferon-? initiated by immune recognition of self nucleic acids. Here we provide first direct evidence that SAMHD1 associates with endogenous nucleic acids in situ. Using fluorescence cross-correlation spectroscopy, we demonstrate that SAMHD1 specifically interacts with ssRNA and ssDNA and establish that nucleic acid-binding and formation of SAMHD1 complexes are mutually dependent. Interaction with nucleic acids and complex formation do not require the SAM domain, but are dependent on the HD domain and the C-terminal region of SAMHD1. We finally demonstrate that mutations associated with AGS exhibit both impaired nucleic acid-binding and complex formation implicating that interaction with nucleic acids is an integral aspect of SAMHD1 function. PMID:23371319

Tüngler, Victoria; Staroske, Wolfgang; Kind, Barbara; Dobrick, Manuela; Kretschmer, Stefanie; Schmidt, Franziska; Krug, Claudia; Lorenz, Mike; Chara, Osvaldo; Schwille, Petra; Lee-Kirsch, Min Ae

2013-06-01

116

Nucleic acid in-situ hybridization detection of infectious agents  

NASA Astrophysics Data System (ADS)

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

Thompson, Curtis T.

2000-04-01

117

Enhanced nucleic acid capture and flow cytometry detection with peptide nucleic acid probes and tunable-surface microparticles  

Microsoft Academic Search

New methods for automated, direct nucleic acid purification and detection are required for the next generation of unattended environmental monitoring devices. In this study we investigated whether tunable-surface bead chemistry and peptide nucleic acids (PNA) could enhance the recovery and detection of intact rRNA in both test tube and automated suspension array hybridization formats. Intact rRNA was easily captured and

Darrell P. Chandler; Ann E. Jarrell

2003-01-01

118

Rapid methods for nucleic acids extraction from Petri dish-grown mycelia  

Microsoft Academic Search

We have developed rapid and economic methods for the isolation of nucleic acids from filamentous fungi. The main advantages of these methods are: (1) the mycelium is directly recovered from a Petri-dish culture, (2) the complete experiment takes place in microfuge tubes, (3) it is very fast and allows for the processing of 24 samples in the same day, and

Gael Lecellier; Philippe Silar

1994-01-01

119

Targeted Delivery of DNA to the Mitochondrial Compartment via Import Sequence-Conjugated Peptide Nucleic Acid  

Microsoft Academic Search

We report that oligonucleotides can be introduced into the mitochondria of living mammalian cells by annealing them to peptide nucleic acids coupled to mitochondrial targeting peptides. These complexes are imported into the mitochondrial matrix through the outer and inner membrane import channels of isolated mitochondria. They are also imported into the mitochondria of cultured cells, provided that the cytosolic uptake

A. Flierl; C. Jackson; B. Cottrell; D. Murdock; P. Seibel; D. C. Wallace

2003-01-01

120

Oxidative Damage to Nucleic Acids in Severe Emphysema*  

PubMed Central

Background Oxidative stress is a key element in the pathogenesis of emphysema, but oxidation of nucleic acids has been largely overlooked. The aim of this study was to investigate oxidative damage to nucleic acids in severe emphysematous lungs. Methods Thirteen human severe emphysematous lungs, including five with ?1-antitrypsin deficiency (AATD), were obtained from patients receiving lung transplantation. Control lung tissue was obtained from non-COPD lungs (n = 8) and donor lungs (n = 8). DNA and RNA oxidation were investigated by immunochemistry. Morphometry (mean linear intercept [Lm] and CT scan) and immunostaining for CD68 and neutrophil elastase also were performed. Results Nucleic acid oxidation was increased in alveolar wall cells in emphysematous lungs compared to non-COPD and donor lungs (p < 0.01). In emphysematous lungs, oxidative damage to nucleic acids in alveolar wall cells was increased in the more severe emphysematous areas assessed by histology (Lm, > 0.5 mm; p < 0.05) and CT scan (< ?950 Hounsfield units; p < 0.05). Compared to classic emphysema, AATD lungs exhibited higher levels of nucleic acid oxidation in macrophages (p < 0.05) and airway epithelial cells (p < 0.01). Pretreatments with DNase and RNase demonstrated that RNA oxidation was more prevalent than DNA oxidation in alveolar wall cells. Conclusions We demonstrated for the first time that nucleic acids, especially RNA, are oxidized in human emphysematous lungs. The correlation between the levels of oxidative damage to nucleic acids in alveolar wall cells and the severity of emphysema suggest a potential role in the pathogenesis of emphysema. PMID:19118262

Deslee, Gaetan; Woods, Jason C.; Moore, Carla; Conradi, Susan H.; Gierada, David S.; Atkinson, Jeffrey J.; Battaile, John T.; Liu, Lucy; Patterson, G. Alexander; Adair-Kirk, Tracy L.; Holtzman, Michael J.; Pierce, Richard A.

2013-01-01

121

A Density Functional Theory Study of the Non-local Correlations between Nucleic Acid Base Pairs  

NASA Astrophysics Data System (ADS)

The interactions of nucleic acid bases are fundamentally important in determining the behavior and structure of biologically important molecules such as DNA and RNA. However, the stacking of nucleic acid bases in a strand of genetic material involves significant van der Waals forces, which are often inaccurately represented or too expensive to compute in many modern theoretical methods. In this paper, we use Density Functional Theory (DFT) with a non-local van der Waals correlation functional to study the stacking interactions of nucleic acid base pairs. This method correctly and seamlessly accounts for the long-range interactions present among isolated fragments through a density-density interaction formula. Since this technique is implemented within DFT it has the advantage of being able to draw on the speed, efficiency and accuracy of this ab initio method. M. Dion, H. Rydberg, E. Schröder, D. C. Langreth and B. I. Lundqvist, Phys. Rev. Lett. 92, 24601-1 (2004).

Cooper, Valentino R.; Thonhauser, Timo; Langreth, David C.

2006-03-01

122

Method for nucleic acid hybridization using single-stranded DNA binding protein  

DOEpatents

Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe. The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample (potentially including the nucleic acid sequence) with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution (i.e., one in which the t.sub.1/2 of renaturation is longer than 3 weeks) of single-stranded DNA greater than 500 fold (i.e., to a t.sub.1/2 less than 60 min, preferably less than 5 min, and most preferably about 1 min.) in the absence of nucleotide triphosphates.

Tabor, Stanley (Cambridge, MA); Richardson, Charles C. (Chestnut Hill, MA)

1996-01-01

123

Conformational transitions in human translin enable nucleic acid binding  

PubMed Central

Translin is a highly conserved RNA- and DNA-binding protein that plays essential roles in eukaryotic cells. Human translin functions as an octamer, but in the octameric crystallographic structure, the residues responsible for nucleic acid binding are not accessible. Moreover, electron microscopy data reveal very different octameric configurations. Consequently, the functional assembly and the mechanism of nucleic acid binding by the protein remain unclear. Here, we present an integrative study combining small-angle X-ray scattering (SAXS), site-directed mutagenesis, biochemical analysis and computational techniques to address these questions. Our data indicate a significant conformational heterogeneity for translin in solution, formed by a lesser-populated compact octameric state resembling the previously solved X-ray structure, and a highly populated open octameric state that had not been previously identified. On the other hand, our SAXS data and computational analyses of translin in complex with the RNA oligonucleotide (GU)12 show that the internal cavity found in the octameric assemblies can accommodate different nucleic acid conformations. According to this model, the nucleic acid binding residues become accessible for binding, which facilitates the entrance of the nucleic acids into the cavity. Our data thus provide a structural basis for the functions that translin performs in RNA metabolism and transport. PMID:23980029

Perez-Cano, Laura; Eliahoo, Elad; Lasker, Keren; Wolfson, Haim J.; Glaser, Fabian; Manor, Haim; Bernado, Pau; Fernandez-Recio, Juan

2013-01-01

124

TOPICAL REVIEW: Nanopore sensors for nucleic acid analysis  

Microsoft Academic Search

In the past decade, nanometre-scale pores have been explored as the basis for technologies to analyse and sequence single nucleic acid molecules. Most approaches involve using such a pore to localize single macromolecules and interact with them to garner some information on their composition. Though nanopore sensors cannot yet claim success at deoxyribonucleic acid (DNA) sequencing, nanopore-based technologies offer one

Jonathan J. Nakane; Mark Akeson; Andre Marziali

2003-01-01

125

Interaction of resveratrol and genistein with nucleic acids.  

PubMed

Resveratrol (RES) and genistein (GEN) are the dietary natural products known to possess chemopreventive property and also the ability to repair DNA damage induced by mutagens/carcinogens. It is believed that the therapeutic activity of these compounds could be primarily due to their interaction with nucleic acids but detailed reports are not available. We here explore the interaction of these drugs with nucleic acids considering DNA and RNA as a potential therapeutic target. The interaction of RES and GEN has been analysed in buffered solution with DNA [saline sodium citrate (SSC)] and RNA [tris ethylene diammine tetra acetic acid (TE)] using UV-absorption and Fourier transform infrared (FTIR) spectroscopy. The UV analysis revealed lesser binding affinity with nucleic acids at lower concentration of RES (P/D = 5.00 and 10.00), while at higher drug concentration (P/D = 0.75, 1.00 and 2.50) hyperchromic effect with shift in the lambda(max) is noted for DNA and RNA. A major RES-nucleic acids complexes was observed through base pairs and phosphate backbone groups with K = 35.782 M(-1) and K = 34.25 M(-1) for DNARES and RNA-RES complexes respectively. At various concentrations of GEN (P/D = 0.25, 0.50, 0.75, 1.00 and 2.50) hyperchromicity with shift in the lambda(max) from 260-->263 nm and 260--> 270 nm is observed for DNA-GEN and RNA-GEN complexes respectively. The binding constant (from UV analysis) for GEN-nucleic acids complexes could not be obtained due to GEN absorbance overlap with that of nucleic acids at 260 nm. Nevertheless a detailed analysis with regard to the interaction of these drugs (RES/GEN) with DNA and RNA could feasibly be understood by FTIR. PMID:15826497

Usha, Subbiah; Johnson, Irudayam Maria; Malathi, Raghunathan

2005-03-31

126

Nucleic acids for the rational design of reaction circuits.  

PubMed

Nucleic acid-based circuits are rationally designed in vitro assemblies that can perform complex preencoded programs. They can be used to mimic in silico computations. Recent works emphasized the modularity and robustness of these circuits, which allow their scaling-up. Another new development has led to dynamic, time-responsive systems that can display emergent behaviors like oscillations. These are closely related to biological architectures and provide an in vitro model of in vivo information processing. Nucleic acid circuits have already been used to handle various processes for technological or biotechnological purposes. Future applications of these chemical smart systems will benefit from the rapidly growing ability to design, construct, and model nucleic acid circuits of increasing size. PMID:23265857

Padirac, Adrien; Fujii, Teruo; Rondelez, Yannick

2013-08-01

127

Point-of-care nucleic acid testing for infectious diseases  

PubMed Central

Nucleic acid testing for infectious diseases at the point of care is beginning to enter clinical practice in developed and developing countries; especially for applications requiring fast turnaround times, and in settings where a centralized laboratory approach faces limitations. Current systems for clinical diagnostic applications are mainly PCR-based, can only be used in hospitals, and are still relatively complex and expensive. Integrating sample preparation with nucleic acid amplification and detection in a cost-effective, robust, and user-friendly format remains challenging. This review describes recent technical advances that might be able to address these limitations, with a focus on isothermal nucleic acid amplification methods. It briefly discusses selected applications related to the diagnosis and management of tuberculosis, HIV, and perinatal and nosocomial infections. PMID:21377748

Niemz, Angelika; Ferguson, Tanya M.; Boyle, David S.

2013-01-01

128

Analysis of single nucleic acid molecules with protein nanopores  

PubMed Central

We describe the methods used in our laboratory for the analysis of single nucleic acid molecules with protein nanopores. The technical section is preceded by a review of the variety of experiments that can be done with protein nanopores. The end goal of much of this work is single-molecule DNA sequencing, although sequencing is not discussed explicitly here. The technical section covers the equipment required for nucleic acid analysis, the preparation and storage of the necessary materials, and aspects of signal processing and data analysis. PMID:20627172

Maglia, Giovanni; Heron, Andrew J.; Stoddart, David; Japrung, Deanpen; Bayley, Hagan

2011-01-01

129

Silanized nucleic acids: a general platform for DNA immobilization.  

PubMed

We have developed a method for simultaneous deposition and covalent cross-linking of oligonucleotide or PCR products on unmodified glass surfaces. By covalently conjugating an active silyl moiety onto oligonucleotides or cDNA in solutions we have generated a new class of modified nucleic acids, namely silanized nucleic acids. Such silanized molecules can be immobilized instantly onto glass surfaces after manual or automated deposition. This method provides a simple and rapid, yet very efficient, solution to the immobilization of prefabricated oligonucleotides and DNA for chip production. PMID:10908345

Kumar, A; Larsson, O; Parodi, D; Liang, Z

2000-07-15

130

Conformational Flexibility of Pyrimidine Ring in Nucleic Acid Bases  

NASA Astrophysics Data System (ADS)

Nucleic acid bases (NABs) have been considered for many years to be planar and conformationally rigid. However, recently, two possible sources of nucleobases nonplanarity have been found. Ab initio quantum-chemical calculations using large basis sets augmented by inclusion of electron correlation and recent experimental studies revealed that amino groups in isolated cytosine, guanine, and adenine adopt a nonplanar trigonal-pyramidal configuration. Since the values of amino group inversion barriers do not exceed approximately 1 kcal mol-1, this group possesses rather flexible geometry. A different source of nonplanarity of nucleobases originates from the high deformability of the pyrimidine ring. Transition of such a ring in uracil, thymine, cytosine, and guanine molecules from a planar equilibrium conformation to a sofa configuration characterized by a relevant torsion angle of ±20° entails an increase of energy by less than 1.5 kcal mol-1. Therefore, at room temperature, certain fraction of isolated DNA bases should possess nonplanar structure of the heterocyclic ring. This review summarizes recent theoretical studies on the flexibility of the NABs.

Shishkin, Oleg V.; Gorb, Leonid; Leszczynski, Jerzy

131

Multicenter Evaluation of the Verigene Clostridium difficile Nucleic Acid Assay  

PubMed Central

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 (? 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 ? 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 027, and all 59 possessed ? 117 in tcdC. Time to results was approximately 2.5 h per specimen. The Verigene CDF test is a novel nucleic acid microarray that reliably detects both C. difficile toxins A and B in unformed stool specimens and appears to adequately identify ribotype 027 isolates. PMID:24088862

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.

2013-01-01

132

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

DOEpatents

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

Nasarabadi, Shanavaz (Livermore, CA)

2011-01-11

133

Nucleotide Composition of Nucleic Acids of Fungi I. Ribonucleic Acids  

PubMed Central

Storck, Roger (The University of Texas, Austin). Nucleotide composition of nucleic acids from fungi. I. Ribonucleic acids. J. Bacteriol. 90:1260–1264. 1965.—The nucleotide composition of the ribonucleic acids (RNA) present in extracts of 26 species of fungi was determined. The results were analyzed, together with those in the literature. It was found that the content in moles per cent of guanine plus cytosine (GC content) varied from 44.1 to 60.5 in a distribution composed of 8 species of zygomycetes, 10 of ascomycetes, 11 of deuteromycetes, and 8 of basidiomycetes. The GC-content range and average were, respectively, 44.1 to 49.3 and 46.4 for the zygomycetes, 47.4 to 54.4 and 50.2 for the ascomycetes, 48.2 to 54.5 and 51.6 for the deuteromycetes, and 50.4 to 60.5 and 52.4 for the basidiomycetes. The GC content averaged 45.6 and ranged from 44.1 to 46.3 for four Mucor species. In addition, GC contents significantly lower than 50 were also encountered in some species of Hemiascomycetidae, suggesting that AT type RNA is not uncommon in fungi. It was proposed that the base composition of fungal RNA might have a taxonomic and phylogenetic significance. PMID:5848326

Storck, Roger

1965-01-01

134

The application of nucleic acid vaccines in veterinary medicine  

Microsoft Academic Search

Nucleic acid immunisation entails the delivery of DNA (or RNA) encoding a vaccine antigen to the recipient. The DNA is taken up by host cells and transcribed to mRNA, from which the vaccine proteins are then translated. The expressed proteins are recognised as foreign by the host immune system and elicit an immune response, which may have both cell-mediated and

Stephen P Dunham

2002-01-01

135

Parsing nucleic acid pseudoknotted secondary structure: algorithm and applications  

E-print Network

Nucleic acids - that is, DNA and RNA molecules - play fundamental roles in the cell: in translation based on dynamic programming aim to find a structure with minimum free energy according to some to calculate the free en- ergy of a pseudoknotted secondary structure. This is useful for heuristic prediction

Condon, Anne

136

Nucleic acid encoding TGF-. beta. and its uses  

SciTech Connect

This patent describes a method. It comprises: constructing a vector which includes nucleic acid encoding biologically active TGF-{beta}, transforming a host eukaryotic cell with the vector, culturing the transformed cell and recovering mature TGF-{beta} from the culture medium.

Derynck, R.M.A.; Goeddel, D.V.

1989-12-12

137

Distinguishing Single-and Double-Stranded Nucleic Acid Molecules  

E-print Network

stretching of these highly flexible molecules. This striking sensitivity to relatively small differencesDistinguishing Single- and Double-Stranded Nucleic Acid Molecules Using Solid-State Nanopores Gary translocated RNA molecules through solid-state nanopores, comparing the signatures of translocating double

Dekker, Nynke

138

Structure, stability and behaviour of nucleic acids in ionic liquids  

PubMed Central

Nucleic acids have become a powerful tool in nanotechnology because of their conformational polymorphism. However, lack of a medium in which nucleic acid structures exhibit long-term stability has been a bottleneck. Ionic liquids (ILs) are potential solvents in the nanotechnology field. Hydrated ILs, such as choline dihydrogen phosphate (choline dhp) and deep eutectic solvent (DES) prepared from choline chloride and urea, are ‘green’ solvents that ensure long-term stability of biomolecules. An understanding of the behaviour of nucleic acids in hydrated ILs is necessary for developing DNA materials. We here review current knowledge about the structures and stabilities of nucleic acids in choline dhp and DES. Interestingly, in choline dhp, A–T base pairs are more stable than G–C base pairs, the reverse of the situation in buffered NaCl solution. Moreover, DNA triplex formation is markedly stabilized in hydrated ILs compared with aqueous solution. In choline dhp, the stability of Hoogsteen base pairs is comparable to that of Watson–Crick base pairs. Moreover, the parallel form of the G-quadruplex is stabilized in DES compared with aqueous solution. The behaviours of various DNA molecules in ILs detailed here should be useful for designing oligonucleotides for the development of nanomaterials and nanodevices. PMID:25013178

Tateishi-Karimata, Hisae; Sugimoto, Naoki

2014-01-01

139

Proficiency of Nucleic Acid Tests for Avian Influenza Viruses, Australasia  

PubMed Central

An avian influenza quality assurance program was used to provide information for laboratories on the sensitivity and specificity of their avian influenza nucleic acid testing. Most laboratories were able to correctly detect clinically relevant amounts of influenza virus (H5N1), and results improved as each subsequent panel was tested. PMID:18598638

Stelzer-Braid, Sacha; Escott, Ros; Baleriola, Cristina; Kirkland, Peter; Robertson, Peter; Catton, Michael

2008-01-01

140

Interactions of calix[n]arenes with nucleic acids.  

PubMed

DNA interaction with artificial binders is of great interest, especially in light of the broad range of possible biomedical applications. The growing understanding of replication, transcription and translation opened the path for new approaches to target pathological effects at a very early stage. Meanwhile, the competitive binding to nucleic acids by designed molecules, which, for example, block certain sequences for natural binders, such as transcription factors, has become a promising concept in the context of gene therapy. On the other extreme, the transport of nucleic acids over the cell membrane into the nucleus by transfection agents opens the possibility to reprogram protein biosynthesis within host cells. In the past decades several substance classes have been developed for a noncovalent specific DNA binding with predictable biological effects, such as peptide nucleic acids or polyamide ligands. Calixarenes have not received so much attention, although they consist of a compact aromatic core tuneable in size, and allow the introduction of cationic functionalities at their upper and lower rims. Formerly being utilized as receptor moieties due to the possibility of complexating guests in their cavities, calixarenes are now also used as molecular scaffolds for multivalent ligands and are, therefore, suitable tools for cooperative DNA complexation. This review surveys specific supramolecular interactions between calixarene derivatives and nucleic acids, with an emphasis on structural elements in the calixarenes and the biological consequences of their complex formation with DNA strands. PMID:22545418

Peters, Max Sena; Li, Miao; Schrader, Thomas

2012-03-01

141

Nucleic acid amplification: Alternative methods of polymerase chain reaction  

PubMed Central

Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically. PMID:24302831

Fakruddin, Md; Mannan, Khanjada Shahnewaj Bin; Chowdhury, Abhijit; Mazumdar, Reaz Mohammad; Hossain, Md. Nur; Islam, Sumaiya; Chowdhury, Md. Alimuddin

2013-01-01

142

Past, present and future of nucleic acids electrochemistry  

Microsoft Academic Search

Electrochemistry of nucleic acids was discovered about 40 years ago. During the first 15 years electrochemistry brought early evidence of DNA premelting and polymorphy of the DNA double helix. At present electrochemical methods working with stationary electrodes are able to detect DNA at attomol and in some cases, even at lower levels. A great progress in the development of electrochemical

Emil Pale?ek

2002-01-01

143

Liver cell specific targeting of peptide nucleic acid oligomers  

Microsoft Academic Search

Chimeric molecules consisting of peptide nucleic acid (PNA) and lactose have been synthesized to test the hypothesis that lactose moieties can promote cell-specific uptake of PNAs. We find that lactose modified PNAs rapidly enter liver-derived HepG2 cells while unmodified PNAs do not and that lactose modified PNAs can inhibit cellular telomerase.

Xiao Zhang; Carla G Simmons; David R Corey

2001-01-01

144

Synthesis, Analysis, Purification, and Intracellular Delivery of Peptide Nucleic Acids  

Microsoft Academic Search

Peptide nucleic acids (PNAs) are nonionic DNA mimics. Their novel chemical properties may facilitate the development of selective and potent antisense and antigene strategies for regulating intracellular processes. Described herein are procedures for the synthesis, purification, handling, and characterization of PNAs. A simple protocol for the lipid-mediated introduction of PNAs into in vitro cultures of mammalian cells is provided.

Dwaine A. Braasch; David R. Corey

2001-01-01

145

Sequence and Structural Selectivity of Nucleic Acid Binding Ligands †  

Microsoft Academic Search

The sequence and structural selectivity of 15 different DNA binding agents was explored using a novel, thermodynamically rigorous, competition dialysis procedure. In the competition dialysis method, 13 different nucleic acid structures were dialyzed against a common ligand solution. More ligand accumulated in the dialysis tube containing the structural form with the highest ligand binding affinity. DNA structural forms included in

Jinsong Ren; Jonathan B. Chaires

1999-01-01

146

Targeting DNA G-Quadruplex Structures with Peptide Nucleic Acids  

PubMed Central

Regulation of genetic functions based on targeting DNA or RNA sequences with complementary oligonucleotides is especially attractive in the post-genome era. Oligonucleotides can be rationally designed to bind their targets based on simple nucleic acid base pairing rules. However, the use of natural DNA and RNA oligonucleotides as targeting probes can cause numerous off-target effects. In addition, natural nucleic acids are prone to degradation in vivo by various nucleases. To address these problems, nucleic acid mimics such as peptide nucleic acids (PNA) have been developed. They are more stable, show less off-target effects, and, in general, have better binding affinity to their targets. However, their high affinity to DNA can reduce their sequence-specificity. The formation of alternative DNA secondary structures, such as the G-quadruplex, provides an extra level of specificity as targets for PNA oligomers. PNA probes can target the loops of G-quadruplex, invade the core by forming PNA-DNA guanine-tetrads, or bind to the open bases on the complementary cytosine-rich strand. Not only could the development of such G-quadruplex-specific probes allow regulation of gene expression, but it will also provide a means to clarify the biological roles G-quadruplex structures may possess. PMID:22376112

Panyutin, Igor G.; Onyshchenko, Mykola I.; Englund, Ethan A.; Appella, Daniel H.; Neumann, Ronald D.

2012-01-01

147

Mosaic protein and nucleic acid vaccines against hepatitis C virus  

DOEpatents

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.

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

2013-06-11

148

Opening of nucleic-acid double strands by helicases: Active versus passive opening M. D. Betterton  

E-print Network

-stranded nucleic-acid NA molecules. The strand separation is fueled by nucleotide triphosphate NTP hydrolysisOpening of nucleic-acid double strands by helicases: Active versus passive opening M. D. Betterton move along double-stranded nucleic-acid molecules and unwind the double helix. This paper presents

Jülicher, Frank

149

1 Optimization of Liganded Polyethylenimine Polyethylene Glycol 2 Vector for Nucleic Acid Delivery  

E-print Network

1 Optimization of Liganded Polyethylenimine Polyethylene Glycol 2 Vector for Nucleic Acid Delivery: The delivery of nucleic acids into cells is an attractive approach for cancer therapy. Polyethylenimine (PEI and cationic lipids, which bind and 26 condense nucleic acids. These nonviral cationic vectors possess 27 many

Lebendiker, Mario

150

A Simple Glycol Nucleic Acid Lilu Zhang, Adam Peritz, and Eric Meggers*  

E-print Network

A Simple Glycol Nucleic Acid Lilu Zhang, Adam Peritz, and Eric Meggers* Department of Chemistry. The discovered glycol nucleic acid (GNA) uses the canonical Watson-Crick base pairing scheme combined 10, 2004; E-mail: meggers@sas.upenn.edu We here wish to disclose a novel nucleic acid analogue which

Meggers, Eric

151

Bifacial Peptide Nucleic Acid as an Allosteric Switch for Aptamer and Ribozyme Function  

E-print Network

Bifacial Peptide Nucleic Acid as an Allosteric Switch for Aptamer and Ribozyme Function Xin Xia demonstrate herein that bifacial peptide nucleic acid (bPNA) hybrid triplexes functionally sub- stitute class of bifacial1 -peptide nucleic acids (bPNAs),2 derived from studies on artificial recognition.3

Bong, Dennis

152

Bifacial Peptide Nucleic Acid Directs Cooperative Folding and Assembly of Binary, Ternary, and Quaternary DNA Complexes  

E-print Network

Bifacial Peptide Nucleic Acid Directs Cooperative Folding and Assembly of Binary, Ternary, which we term bifacial peptide nucleic acid (bPNA), function as a noncovalent template for thymine are brought together on a bridging bifacial melamine- displaying peptide nucleic acid (bPNA) template strand

Bong, Dennis

153

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

E-print Network

DNA-Templated Polymerization of Side-Chain-Functionalized Peptide Nucleic Acid Aldehydes Ralph E previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid-specific DNA-templated polymerization of unfunc- tionalized peptide nucleic acid (PNA) aldehydes using

Liu, David R.

154

Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future  

Microsoft Academic Search

Synthetic molecules that can bind with high sequence specificity to a chosen target in a gene sequence are of major interest in medicinal and biotechnological contexts. They show promise for the development of gene therapeutic agents, diag- nostic devices for genetic analysis, and as molecular tools for nucleic acid manipulations. Peptide nucleic acid (PNA) is a nucleic acid analog in

ARGHYA RAY; BENGT NORDEN

155

Highly Cooperative Behavior of Peptide Nucleic Acid-Linked DNA-Modified Gold-Nanoparticle and  

E-print Network

Highly Cooperative Behavior of Peptide Nucleic Acid-Linked DNA-Modified Gold-Nanoparticle and Comb whether particle aggregates can be held together with peptide nucleic acids (PNAs),[15,16] uncharged of nucleic acids, proteins, metal ions, and small molecules.[1­10] When complementary mixtures

156

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

E-print Network

Templated Synthesis of Peptide Nucleic Acids via Sequence-Selective Base-Filling Reactions Jennifer describe the templated synthesis of both native and modified peptide nucleic acids (PNAs) through base, 2009; E-mail: drliu@fas.harvard.edu Template-directed nucleic acid synthesis is an essential compo

Liu, David R.

157

Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology  

Microsoft Academic Search

During the past ten years, the DNA mimic peptide nucleic acid has inspired the development of a variety of hybridisation-based methods for detection, quantification, purification and characterisation of nucleic acids. Most of these methods have taken advantage of the very favourable DNA and RNA hybridisation properties of peptide nucleic acids combined with the unique properties and opportunities offered by peptide

Peter E Nielsen

2001-01-01

158

A Theoretical Analysis of Specificity of Nucleic Acid Interactions with Oligonucleotides and Peptide  

E-print Network

and Peptide Nucleic Acids (PNAs) Aleksey Lomakin1 and Maxim D. Frank-Kamenetskii2 * 1 Physics Department analog or its mimic (such as peptide nucleic acid, or PNA). We consider simplest models with onlyA Theoretical Analysis of Specificity of Nucleic Acid Interactions with Oligonucleotides

Benedek, George B.

159

Peptide nucleic acid probes with charged photocleavable mass markers  

PubMed Central

Halogen-labelled peptide organic acid (HPOA) monomers have been synthesised and incorporated into sequence-specific peptide nucleic acid (PNA) probes. Three different types of probe have been prepared; the unmodified PNA probe, the PNA probe with a mass marker, and the PNA probe with photocleavable mass marker. All three types of probe have been used in model studies to develop a mass spectrometry-based hybridisation assay for detection of point mutations in DNA. PMID:21687524

Ball, Rachel J; Green, Philip S; Gale, Nittaya; Langley, G John

2010-01-01

160

Binding Properties in Protein Nucleic Acids  

E-print Network

tor for the stability of base paired systems and for the selectivity of the pairing. In the gas-phase, it has been indeed found experimentally and by calculations, that each hydrogen bond has a strength of 5-7 kcal/mol 2, 3 . In aqueous medium, however, a hydrogen bond contributes only 0.5-1.8 kcal/mol 4--6 . The weakness of hydrogen bonds in water results because the 63 N N N O R H N N O N R H H N H H Figure 2. Guanine-cytosine-pair in DNA N N N N R N N O O R H H CH 3 H Figure 3. Adenine-thymine-pair in DNA nucleic bases form strong bonds to themselves and to water molecules. Van-der-Waals and dipole interaction between the planar base pairs lead to the so-called #- or base stacking interaction which is the second decisive interaction enabling the formation of duplexes 7 . Van-der-Waals interaction, that is caused by fluctuating dipols, seems to play a central role for this stabilizing factor. In addition, permanent electrostatic attractions and hy

Horst Rollnik; T. A. Hupp; Dietrich Wolf (editors; B. Dietrich; B. Dietrich; B. Engels; B. Engels

2001-01-01

161

Insights on the role of nucleic acid/protein interactions in chaperoned nucleic acid rearrangements of HIV-1 reverse transcription  

PubMed Central

HIV-1 reverse transcription requires several nucleic acid rearrangement steps that are “chaperoned” by the nucleocapsid protein (NC), including minus-strand transfer, in which the DNA transactivation response element (TAR) is annealed to the complementary TAR RNA region of the viral genome. These various rearrangement processes occur in NC bound complexes of specific RNA and DNA structures. A major barrier to the investigation of these processes in vitro has been the diversity and heterogeneity of the observed nucleic acid/protein assemblies, ranging from small complexes of only one or two nucleic acid molecules all the way up to large-scale aggregates comprised of thousands of NC and nucleic acid molecules. Herein, we use a flow chamber approach involving rapid NC/nucleic acid mixing to substantially control aggregation for the NC chaperoned irreversible annealing kinetics of a model TAR DNA hairpin sequence to the complementary TAR RNA hairpin, i.e., to form an extended duplex. By combining the flow chamber approach with a broad array of fluorescence single-molecule spectroscopy (SMS) tools (FRET, molecule counting, and correlation spectroscopy), we have unraveled the complex, heterogeneous kinetics that occur during the course of annealing. The SMS results demonstrate that the TAR hairpin reactant is predominantly a single hairpin coated by multiple NCs with a dynamic secondary structure, involving equilibrium between a “Y” shaped conformation and a closed one. The data further indicate that the nucleation of annealing occurs in an encounter complex that is formed by two hairpins with one or both of the hairpins in the “Y” conformation. PMID:17372205

Liu, Hsiao-Wei; Zeng, Yining; Landes, Christy F.; Kim, Yoen Joo; Zhu, Yongjin; Ma, Xiaojing; Vo, My-Nuong; Musier-Forsyth, Karin; Barbara, Paul F.

2007-01-01

162

Nucleic Acid and Non-Nucleic Acid-Based Reprogramming of Adult Limbal Progenitors to Pluripotency  

PubMed Central

Reprogramming somatic cells to a pluripotent state by nucleic acid based (NAB) approaches, involving the ectopic expression of transcription factors, has emerged as a standard method. We recently demonstrated that limbal progenitors that regenerate cornea are reprogrammable to pluripotency by a non-NAB approach through simple manipulation of microenvironment thus extending the possible therapeutic use of these readily accessible cells beyond the proven treatment of corneal diseases and injury. Therefore, to determine the validity and robustness of non-cell autonomous reprogramming of limbal progenitors for a wider clinical use, here, we have compared their reprogramming by non-NAB and NAB approaches. We observed that both approaches led to (1) the emergence of colonies displaying pluripotency markers, accompanied by a temporal reciprocal changes in limbal-specific and pluripotency gene expression, and (2) epigenetic alterations of Oct4 and Nanog, associated with the de-novo activation of their expression. While the efficiency of reprogramming and passaging of re-programmed cells were significantly better with the NAB approach, the non-NAB approach, in contrast, led to a regulated reprogramming of gene expression, and a significant decrease in the expression of Hormad1, a gene associated with immunogenic responses. The reprogramming efficiency by non-NAB approach was influenced by exosomes present in conditioned medium. Cells reprogrammed by both approaches were capable of differentiating along the three germ lineages and generating chimeras. The analysis suggests that both approaches are effective in reprogramming limbal progenitors but the non-NAB approach may be more suitable for potential clinical applications by averting the risk of insertional mutagenesis and immune responses associated with the NAB approach. PMID:23056428

Parameswaran, Sowmya; Balasubramanian, Sudha; Babai, Norbert; DelDebbio, Carolina B.; Harms, Donald W.; Gurumurthy, Channabasavaiah B.; Rao, Mahendra S.; Sharp, John G.; Ahmad, Iqbal

2012-01-01

163

The use of Sonogashira coupling for the synthesis of modified uracil peptide nucleic acid  

Microsoft Academic Search

Palladium-catalyzed Sonogashira coupling has been shown to be compatible with PNA monomers as illustrated by the reaction of 5-iodouracil peptide nucleic acid monomer (IU-PNA) with several terminal alkynes. These reactions have been performed in the solution phase and with IU-PNA linked to an insoluble polymer support. The results presented herein show that while the isolated yields from the solution phase

Robert H. E Hudson; Ge Li; Joseph Tse

2002-01-01

164

Nucleic acids encoding modified human immunodeficiency virus type 1 (HIV-1) group M consensus envelope glycoproteins  

SciTech Connect

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.

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

165

Multicenter Evaluation of a Candida albicans Peptide Nucleic Acid Fluorescent In Situ Hybridization Probe for Characterization of Yeast Isolates from Blood Cultures  

Microsoft Academic Search

Yeasts, long known to cause thrush and vulvovaginal infec- tions, have emerged as a significant cause of nosocomial infec- tions (18, 21). Although Candida albicans usually is the most frequently isolated organism from patients with fungemia, other species of Candida are also important pathogens in hos- pitalized patients (18, 21). Furthermore, some of the Candida species other than C. albicans

D. A. Wilson; M. J. Joyce; L. S. Hall; L. B. Reller; G. D. Roberts; G. S. Hall; B. D. Alexander; G. W. Procop

2005-01-01

166

Synthesis of modified peptide nucleic acids.  

E-print Network

??Les Acides Peptido-nucléiques (PNA) sont des analogues synthétiques d’oligonucleotides naturels, ils sont constitués d’une répétition d’unités N-(2-aminoethyl)-glycine reliées par une liaison peptidique. Leur stabilité chimique… (more)

Chouikhi, Dalila

2013-01-01

167

Ultrasensitive nucleic acid sequence detection by single-molecule electrophoresis  

SciTech Connect

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

Castro, A; Shera, E.B.

1996-09-01

168

A partition function algorithm for interacting nucleic acid strands  

Microsoft Academic Search

Recent interests, such as RNA interference and antisense RNA regulation, strongly motivate the problem of predicting whether two nucleic acid strands interact. Motivation: Regulatory non-coding RNAs (ncRNAs) such as microRNAs play an important role in gene regulation. Studies on both prokaryotic and eukaryotic cells show that such ncRNAs usually bind to their target mRNA to regulate the translation of corresponding

Hamidreza Chitsaz; Raheleh Salari; Süleyman Cenk Sahinalp; Rolf Backofen

2009-01-01

169

System for portable nucleic acid testing in low resource settings  

NASA Astrophysics Data System (ADS)

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.

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

2013-03-01

170

Nucleic acid oxidation: an early feature of Alzheimer's disease.  

PubMed

Studies of oxidative damage during the progression of Alzheimer's disease (AD) suggest its central role in disease pathogenesis. To investigate levels of nucleic acid oxidation in both early and late stages of AD, levels of multiple base adducts were quantified in nuclear and mitochondrial DNA from the superior and middle temporal gyri (SMTG), inferior parietal lobule (IPL), and cerebellum (CER) of age-matched normal control subjects, subjects with mild cognitive impairment, preclinical AD, late-stage AD, and non-AD neurological disorders (diseased control; DC) using gas chromatography/mass spectrometry. Median levels of multiple DNA adducts in nuclear and mitochondrial DNA were significantly (p ? 0.05) elevated in the SMTG, IPL, and CER in multiple stages of AD and in DC subjects. Elevated levels of fapyguanine and fapyadenine in mitochondrial DNA suggest a hypoxic environment early in the progression of AD and in DC subjects. Overall, these data suggest that oxidative damage is an early event not only in the pathogenesis of AD but is also present in neurodegenerative diseases in general. Levels of oxidized nucleic acids in nDNA and mtDNA were found to be significantly elevated in mild cognitive impairment (MCI), preclinical Alzheimer's disease (PCAD), late-stage AD (LAD), and a pooled diseased control group (DC) of frontotemporal dementia (FTD) and dementia with Lewy bodies (DLB) subjects compared to normal control (NC) subjects. Nucleic acid oxidation peaked early in disease progression and remained elevated. The study suggests nucleic acid oxidation is a general event in neurodegeneration. PMID:24032632

Bradley-Whitman, Melissa A; Timmons, Michael D; Beckett, Tina L; Murphy, Michael P; Lynn, Bert C; Lovell, Mark A

2014-01-01

171

Developing nucleic acid-based electrical detection systems  

Microsoft Academic Search

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

Magdalena Gabig-Ciminska

2006-01-01

172

Inhibition of a DNA-helicase by peptide nucleic acids  

Microsoft Academic Search

Bis-peptide nucleic acid (bis-PNA) binding results in D-loop formation by strand displacement at comple- mentary homopurine stretches in DNA duplexes. Transcription and replication in intact cells is mediated by multienzymatic complexes involving several proteins other than polymerases. The behaviour of the highly stable clamp structure formed by bis-PNAs has thus far been studied with respect to their capacity to arrest

Lionel Bastide; Paul E. Boehmer; Giuseppe Villani; Bernard Lebleu

1999-01-01

173

Inhibiting transcription of chromosomal DNA with antigene peptide nucleic acids  

Microsoft Academic Search

Synthetic molecules that recognize specific sequences within cellular DNA are potentially powerful tools for investigating chromosome structure and function. Here, we designed antigene peptide nucleic acids (agPNAs) to target the transcriptional start sites for the human progesterone receptor B (hPR-B) and A (hPR-A) isoforms at sequences predicted to be single-stranded within the open complex of chromosomal DNA. We found that

Bethany A Janowski; Kunihiro Kaihatsu; Kenneth E Huffman; Jacob C Schwartz; Rosalyn Ram; Daniel Hardy; Carole R Mendelson; David R Corey

2005-01-01

174

DNA-like double helix formed by peptide nucleic acid  

Microsoft Academic Search

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 acid (PNA)1-7 is a DNA analogue with a backbone consisting of N-(2-aminoethyl)glycine units (Fig.

Pernilla Wittung; Peter E. Nielsen; Ole Buchardt; Michael Egholm; Bengt Nordén

1994-01-01

175

Nucleic acid-based tissue biomarkers of urologic malignancies.  

PubMed

Molecular biomarkers play an important role in the clinical management of cancer patients. Biomarkers allow estimation of the risk of developing cancer; help to diagnose a tumor, ideally at an early stage when cure is still possible; and aid in monitoring disease progression. Furthermore, they hold the potential to predict the outcome of the disease (prognostic biomarkers) and the response to therapy (predictive biomarkers). Altogether, biomarkers will help to avoid tumor-related deaths and reduce overtreatment, and will contribute to increased survival and quality of life in cancer patients due to personalized treatments. It is well established that the process of carcinogenesis is a complex interplay between genomic predisposition, acquired somatic mutations, epigenetic changes and genomic aberrations. Within this complex interplay, nucleic acids, i.e. RNA and DNA, play a fundamental role and therefore represent ideal candidates for biomarkers. They are particularly promising candidates because sequence-specific hybridization and amplification technologies allow highly accurate and sensitive assessment of these biomarker levels over a broad dynamic range. This article provides an overview of nucleic acid-based biomarkers in tissues for the management of urologic malignancies, i.e. tumors of the prostate, testis, kidney, penis, urinary bladder, renal pelvis, ureter and other urinary organs. Special emphasis is put on genomic, transcriptomic and epigenomic biomarkers (SNPs, mutations [genomic and mitochondrial], microsatellite instabilities, viral and bacterial DNA, DNA methylation and hydroxymethylation, mRNA expression, and non-coding RNAs [lncRNA, miRNA, siRNA, piRNA, snRNA, snoRNA]). Due to the multitude of published biomarker candidates, special focus is given to the general applicability of different molecular classes as biomarkers and some particularly promising nucleic acid biomarkers. Furthermore, specific challenges regarding the development and clinical implementation of nucleic acid-based biomarkers are discussed. PMID:24878394

Dietrich, Dimo; Meller, Sebastian; Uhl, Barbara; Ralla, Bernhard; Stephan, Carsten; Jung, Klaus; Ellinger, Jörg; Kristiansen, Glen

2014-08-01

176

Nucleic acid-lipid membrane interactions studied by DSC  

PubMed Central

The interactions of nucleic acids with lipid membranes are of great importance for biological mechanisms as well as for biotechnological applications in gene delivery and drug carriers. The optimization of liposomal vectors for clinical use is absolutely dependent upon the formation mechanisms, the morphology, and the molecular organization of the lipoplexes, that is, the complexes of lipid membranes with DNA. Differential scanning calorimetry (DSC) has emerged as an efficient and relatively easy-to-operate experimental technique that can straightforwardly provide data related to the thermodynamics and the kinetics of the DNA—lipid complexation and especially to the lipid organization and phase transitions within the membrane. In this review, we summarize DSC studies considering nucleic acid—membrane systems, accentuating DSC capabilities, and data analysis. Published work involving cationic, anionic, and zwitterionic lipids as well as lipid mixtures interacting with RNA and DNA of different sizes and conformations are included. It is shown that despite limitations, issues such as DNA- or RNA-induced phase separation and microdomain lipid segregation, liposomal aggregation and fusion, alterations of the lipid long-range molecular order, as well as membrane-induced structural changes of the nucleic acids can be efficiently treated by systematic high-sensitivity DSC studies. PMID:21430956

Giatrellis, Sarantis; Nounesis, George

2011-01-01

177

Molecular Modeling of Nucleic Acid Structure: Electrostatics and Solvation  

PubMed Central

This unit presents an overview of computer simulation techniques as applied to nucleic acid systems, ranging from simple in vacuo molecular modeling techniques to more complete all-atom molecular dynamics treatments that include an explicit representation of the environment. The third in a series of four units, this unit focuses on critical issues in solvation and the treatment of electrostatics. UNITS 7.5 & 7.8 introduced the modeling of nucleic acid structure at the molecular level. This included a discussion of how to generate an initial model, how to evaluate the utility or reliability of a given model, and ultimately how to manipulate this model to better understand the structure, dynamics, and interactions. Subject to an appropriate representation of the energy, such as a specifically parameterized empirical force field, the techniques of minimization and Monte Carlo simulation, as well as molecular dynamics (MD) methods, were introduced as means to sample conformational space for a better understanding of the relevance of a given model. From this discussion, the major limitations with modeling, in general, were highlighted. These are the difficult issues in sampling conformational space effectively—the multiple minima or conformational sampling problems—and accurately representing the underlying energy of interaction. In order to provide a realistic model of the underlying energetics for nucleic acids in their native environments, it is crucial to include some representation of solvation (by water) and also to properly treat the electrostatic interactions. These are discussed in detail in this unit. PMID:18428877

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

2014-01-01

178

Nucleic Acid Sample Preparation using Spontaneous Biphasic Plug Flow  

PubMed Central

Nucleic acid (NA) extraction and purification has become a common technique in both research and clinical laboratories. Current methods require repetitive wash steps with a pipette that is laborious and time consuming making it inefficient for clinical settings. We present here a simple technique that relies on spontaneous biphasic plug flow inside a capillary to achieve sample preparation. By filling the sample with oil, aqueous contaminants were displaced from the captured NA without pipetting wash buffers or use of external force and equipment. mRNA from mammalian cell culture was purified and PCR amplification showed similar threshold cycle values as those obtained from a commercially available kit. HIV viral like particles were spiked into serum and a 5-fold increase in viral RNA extraction yield was achieved compared to the conventional wash method. In addition, viral RNA was successfully purified from human whole blood, and a limit of detection of approximately 14 copies of RNA extracted per sample. The results demonstrate the utility of the current technique for nucleic acid purification for clinical purposes, and the overall approach provides a potential method to implement nucleic acid testing in low resource settings. PMID:23941230

Thomas, Peter C.; Strotman, Lindsay N.; Theberge, Ashleigh B.; Berthier, Erwin; O'Connell, Rachel; Loeb, Jennifer M.; Berry, Scott M.; Beebe, David J.

2013-01-01

179

Detection of group 2a coronaviruses with emphasis on bovine and wild ruminant strains. Virus isolation and detection of antibody, antigen, and nucleic acid.  

PubMed

Group 2a of the Coronaviridae family contains human and animal pathogens that include mouse hepatitis virus, rat coronavirus, human respiratory coronaviruses OC43 and the recently identified HKU1 strain, a newly recognized canine respiratory coronavirus, porcine hemagglutinating encephalomyelitis virus, equine coronavirus, bovine coronavirus (BCoV), and wild-ruminant coronaviruses. The presence of a hemagglutinin-esterase (HE) surface glycoprotein in addition to the viral spike protein is a distinguishing characteristic of most group 2a coronaviruses. BCoV is ubiquitous in cattle worldwide and is an economically significant cause of calf diarrhea, winter dysentery of adult cattle, and respiratory disease in calves and feedlot cattle. We have developed and optimized laboratory diagnostic techniques, including virus isolation in HRT-18 cell cultures, antibody and antigen ELISA, and RT-PCR, for rapid, sensitive, and reliable diagnosis of BCoV and related wild ruminant coronaviruses. PMID:19057864

Hasoksuz, Mustafa; Vlasova, Anastasia; Saif, Linda J

2008-01-01

180

Studies on differentiation of Mûllerian ducts in the quail, Coturnix coturnix japonica. II. Effects of sex hormones on nucleic acid synthesis in isolated female ducts.  

PubMed

The present investigations were carried out in an effort to determine the mechanisms underlying differentiation in the avian Müllerian duct, especially the asymmetrical differentiation of the female ducts. Using the isolated female ducts of Japanese quail, the incorporations of 3H-uridine and 14C-thymidine were determined in vitro at several embryonic stages. Incorporation of 3H-uridine was altered with some synchronous fluctuation during the embryonic period in both the left and right ducts, while 14C-thymidine incorporation first decreased rapidly and subsequently, only slightly, during the same period. By administering sex hormones in vitro nucleotide incorporation was affected to an appreciable extent characteristic of the duct at each stage. This hormonal susceptibility was also periodically altered during duct differentiation. The growing left duct continued to receive an apparent stimulation under the hormonal conditions, while the involuting right duct was sometimes inhibited under the same conditions. Such hormonal susceptibilities may explain the asymmetrical differentiation of the female left and right ducts during this embryonic period. PMID:1127406

Iwamura, Y; Koshihara, H; Noumura, T

1975-04-01

181

Azide-alkyne cycloaddition for universal post-synthetic modifications of nucleic acids and effective synthesis of bioactive nucleic acid conjugates.  

PubMed

The regioselective post-synthetic modifications of nucleic acids are essential to studies of these molecules for science and applications. Here we report a facile universal approach by harnessing versatile phosphoramidation reactions to regioselectively incorporate alkynyl/azido groups into post-synthetic nucleic acids primed with phosphate at the 5' termini. With and without the presence of copper, the modified nucleic acids were subjected to azide-alkyne cycloaddition to afford various nucleic acid conjugates including a peptide-oligonucleotide conjugate (POC) with high yield. The POC was inoculated with human A549 cells and demonstrated excellent cell-penetrating ability despite cell deformation caused by a small amount of residual copper chelated to the POC. The combination of phosphoramidation and azide-alkyne cycloaddition reactions thus provides a universal regioselective strategy to post-synthetically modify nucleic acids. This study also explicated the toxicity of residual copper in synthesized bioconjugates destined for biological systems. PMID:25007778

Su, Yu-Chih; Lo, Yu-Lun; Hwang, Chi-Ching; Wang, Li-Fang; Wu, Min Hui; Wang, Eng-Chi; Wang, Yun-Ming; Wang, Tzu-Pin

2014-09-14

182

Deep Ultraviolet Mapping of Intracellular Protein and Nucleic Acid in Femtograms per Pixel  

PubMed Central

By using imaging spectrophotometry with paired images in the 200- to 280-nm wavelength range, we have directly mapped intracellular nucleic acid and protein distributions across a population of Chinese hamster ovary (CHO-K1) cells. A broadband 100× objective with a numerical aperture of 1.2NA (glycerin immersion) and a novel laser-induced-plasma point source generated high-contrast images with short (~100 ms) exposures and a lateral resolution nearing 200 nm that easily resolves internal organelles. In a population of 420 CHO-K1 cells and 477 nuclei, we found a G1 whole-cell nucleic acid peak at 26.6 pg, a nuclear-isolated total nucleic acid peak at 11.4 pg, and, as inferred by RNase treatment, a G1 total DNA mass of 7.4 pg. At the G1 peak we found a whole-cell protein mass of 95.6 pg, and a nuclear-isolated protein mass of 39.3 pg. An algorithm for protein quantification that senses peptide-bond (220-nm) absorbance was found to have a higher signal-to-noise ratio and to provide more reliable nucleic acid and protein determinations when compared to more classical 280-nm/260-nm algorithms when used for intracellular mass mapping. Using simultaneous imaging with common nuclear stains (Hoechst 33342, Syto-14, and Sytox Orange), we have compared staining patterns to deep-UV images of condensed chromatin and have confirmed bias of these common nuclear stains related to nuclear packaging. The approach allows absolute mass measurements with no special sample preparation or staining. It can be used in conjunction with normal fluorescence microscopy and with relatively modest modification of the microscope. PMID:21796773

Cheung, Man C.; Evans, James G.; McKenna, Brian; Ehrlich, Daniel J.

2011-01-01

183

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

NASA Astrophysics Data System (ADS)

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.

Szeto, Kylan; Craighead, Harold G.

2014-09-01

184

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

NASA Technical Reports Server (NTRS)

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.

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

2011-01-01

185

Microchip Module for Blood Sample Preparation and Nucleic Acid Amplification Reactions  

PubMed Central

A computer numerical control-machined plexiglas-based microchip module was designed and constructed for the integration of blood sample preparation and nucleic acid amplification reactions. The microchip module is comprised of a custom-made heater-cooler for thermal cycling, a series of 254 ?m?×?254 ?m microchannels for transporting human whole blood and reagents in and out of an 8–9 ?L dual-purpose (cell isolation and PCR) glass-silicon microchip. White blood cells were first isolated from a small volume of human whole blood (<3 ?L) in an integrated cell isolation–PCR microchip containing a series of 3.5-?m feature-sized “weir-type” filters, formed by an etched silicon dam spanning the flow chamber. A genomic target, a region in the human coagulation Factor V gene (226-bp), was subsequently directly amplified by microchip-based PCR on DNA released from white blood cells isolated on the filter section of the microchip mounted onto the microchip module. The microchip module provides a convenient means to simplify nucleic acid analyses by integrating two key steps in genetic testing procedures, cell isolation and PCR and promises to be adaptable for additional types of integrated assays. PMID:11230164

Yuen, Po Ki; Kricka, Larry J.; Fortina, Paolo; Panaro, Nicholas J.; Sakazume, Taku; Wilding, Peter

2001-01-01

186

Solution spectroscopy of dipicolinic acid interaction with nucleic acids: Role in spore heat resistance  

Microsoft Academic Search

The effect of dipicolinic acid (DPA) or its calcium chelate (CaDPA) on the spectral characteristics of nucleic acids was examined. Dipicolinic acid was found to displace ethidium gromide from DNA; this indicates that it may bind by intercalation. On interaction with DNA, the ultraviolet absorption spectrum revealed downfield shifts and caused progressive diminution in both DNA and dipicolinate chromophores. The

James A. Lindsay; William G. Murrell

1986-01-01

187

Poly(alkylene oxide) copolymers for nucleic acid delivery.  

PubMed

The advancement of gene-based therapeutics to the clinic is limited by the ability to deliver physiologically relevant doses of nucleic acids to target tissues safely and effectively. Over the last couple of decades, researchers have successfully employed polymer and lipid based nanoassemblies to deliver nucleic acids for the treatment of a variety of diseases. Results of phase I/II clinical studies to evaluate the efficacy and biosafety of these gene delivery vehicles have been encouraging, which has promoted the design of more efficient and biocompatible systems. Research has focused on designing carriers to achieve biocompatibility, stability in the circulatory system, biodistribution to target the disease site, and intracellular delivery, all of which enhance the resulting therapeutic effect. The family of poly(alkylene oxide) (PAO) polymers includes random, block, and branched structures, among which the ABA type triblocks copolymers of ethylene oxide (EO) and propylene oxide (PO) (commercially known as Pluronic) have received the greatest consideration. In this Account, we highlight examples of polycation-PAO conjugates, liposome-PAO formulations, and PAO micelles for nucleic acid delivery. Among the various polymer design considerations, which include molecular weight of polymer, molecular weight of blocks, and length of blocks, the overall hydrophobic-lipophilic balance (HLB) is a critical parameter in defining the behavior of the polymer conjugates for gene delivery. We discuss the effects of varying this parameter in the context of improving gene delivery processes, such as serum stability and association with cell membranes. Other innovative macromolecular modifications discussed in this category include our work to enhance the serum stability and efficiency of lipoplexes using PAO graft copolymers, the development of a PAO gel-based carrier for sustained and stimuli responsive delivery, and the development of biodegradable PAO-based amphiphilic block copolymers. PMID:22260518

Mishra, Swati; Peddada, Lavanya Y; Devore, David I; Roth, Charles M

2012-07-17

188

Interaction of nucleic acids with carbon nanotubes and dendrimers.  

PubMed

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. PMID:22750983

Nandy, Bidisha; Santosh, Mogurampelly; Maiti, Prabal K

2012-07-01

189

On the stability of double stranded nucleic acids.  

PubMed

We present the first pressure-versus-temperature phase diagram for the helix-to-coil transition of double stranded nucleic acids. The thermodynamic stability of a nucleic acid duplex is a complex function of temperature and pressure and strongly depends on the denaturation temperature, T(M), of the duplex at atmospheric pressure. Depending upon T(M), pressure, and temperature, the phase diagram shows that pressure may stabilize, destabilize, or have no effect on the conformational state of DNA. To verify the phase diagram, we have conducted high-pressure UV melting experiments on poly(dIdC)poly(dIdC), a DNA duplex, poly(rA)poly(rU), an RNA duplex, and poly(dA)poly(rU), a DNA/RNA hybrid duplex. The T(M) values of these duplexes have been modulated by altering the solution ionic strength. Significantly, at low salt, these three duplexes have helix-to-coil transition temperatures of 50 degrees C or less. In agreement with the derived phase diagram, we found that the polymeric duplexes were destabilized by pressure if the T(M) is < approximately 50 degrees C. However, these duplexes were stabilized by pressure if the T(M) is > approximately 50 degrees C. The DNA/RNA hybrid duplex, poly(dA)poly(rU), with a T(M) of 31 degrees C in 20 mM NaCl undergoes a pressure-induced helix-to-coil transition at room temperature. This is the first report of pressure-induced denaturation of a nucleic acid duplex and provides new insights into the molecular forces stabilizing these structures. PMID:11562205

Dubins, D N; Lee, A; Macgregor, R B; Chalikian, T V

2001-09-26

190

Poly(alkylene oxide) Copolymers for Nucleic Acid Delivery  

PubMed Central

CONSPECTUS The advancement of gene-based therapeutics to the clinic is limited by the ability to deliver physiologically relevant doses of nucleic acids to target tissues safely and effectively. Polymer and lipid based nano-assemblies have been successfully employed over the last couple of decades for the delivery of nucleic acids to treat a variety of disease states. Results of phase I/II clinical studies to evaluate the efficacy and biosafety of these gene delivery vehicles have been encouraging, thus promoting the design of more efficient and biocompatible systems. Research has focused on designing carriers to achieve biocompatibility, stability in the circulatory system, biodistribution to target the disease site, and intracellular delivery, all of which enhance the resulting therapeutic effect. The family of poly(alkylene oxide) (PAO) includes random, block and branched polymers, among which the ABA type triblocks copolymers of ethylene oxide (EO) and propylene oxide (PO) (commercially known as Pluronic®) have received the greatest consideration. In this Account, we highlight examples of polycation-PAO conjugates, liposome-PAO formulations, and PAO micelles for nucleic acid delivery. Among the various polymer design consideration, which include molecular weight of polymer, molecular weight of blocks, and length of blocks, it has been found that the overall hydrophobic-lipophilic balance (HLB) is a critical parameter in defining the behavior of the polymer conjugates for gene delivery. The effects of varying this parameter are discussed in the context of improving gene delivery processes, such as serum-stability and association with cell membranes. Other innovative macromolecular modifications discussed in this category include the work done by our group to enhance the serum stability and efficiency of lipoplexes using PAO graft copolymers, development of a PAO gel-based carrier for sustained and stimuli responsive delivery, and biodegradable PAO-based amphiphilic block copolymers. PMID:22260518

Mishra, Swati; Peddada, Lavanya Y.; Devore, David I.; Roth, Charles M.

2012-01-01

191

Functional nucleic-Acid-based sensors for environmental monitoring.  

PubMed

Efforts to replace conventional chromatographic methods for environmental monitoring with cheaper and easy to use biosensors for precise detection and estimation of hazardous environmental toxicants, water or air borne pathogens as well as various other chemicals and biologics are gaining momentum. Out of the various types of biosensors classified according to their bio-recognition principle, nucleic-acid-based sensors have shown high potential in terms of cost, sensitivity, and specificity. The discovery of catalytic activities of RNA (ribozymes) and DNA (DNAzymes) which could be triggered by divalent metallic ions paved the way for their extensive use in detection of heavy metal contaminants in environment. This was followed with the invention of small oligonucleotide sequences called aptamers which can fold into specific 3D conformation under suitable conditions after binding to target molecules. Due to their high affinity, specificity, reusability, stability, and non-immunogenicity to vast array of targets like small and macromolecules from organic, inorganic, and biological origin, they can often be exploited as sensors in industrial waste management, pollution control, and environmental toxicology. Further, rational combination of the catalytic activity of DNAzymes and RNAzymes along with the sequence-specific binding ability of aptamers have given rise to the most advanced form of functional nucleic-acid-based sensors called aptazymes. Functional nucleic-acid-based sensors (FNASs) can be conjugated with fluorescent molecules, metallic nanoparticles, or quantum dots to aid in rapid detection of a variety of target molecules by target-induced structure switch (TISS) mode. Although intensive research is being carried out for further improvements of FNAs as sensors, challenges remain in integrating such bio-recognition element with advanced transduction platform to enable its use as a networked analytical system for tailor made analysis of environmental monitoring. PMID:24903959

Sett, Arghya; Das, Suradip; Bora, Utpal

2014-10-01

192

Liquid Chromatography-Mass Spectrometry of Nucleic Acids  

Microsoft Academic Search

\\u000a By virtue of its high-resolving capability, short analysis time, and advanced instrumentation high-performance liquid chromatography\\u000a (HPLC) has become a versatile technique for the separation and characterization of nucleic acids. Among the various chromatographic\\u000a modes, which have been summarized in several reviews (1–6), ion-pair reversed-phase HPLC (IP-RPHPLC) represents the most popular chromatographic technique applicable to the separation\\u000a of single-and double-stranded DNA

Herbert Oberacher; Walther Parson

193

[Mass spectrometry of nucleic acids in molecular medicine].  

PubMed

A stable streamlining trend in the field of medical diagnostics by practical adoption of high-tech and knowledge-intensive analytical systems providing for molecular level studies has appeared during the last few decades. An illustrative example of such technologies is mass spectrometry methods for analyzing biomolecules. This review is intended to brief the potential of the state-of-the-art inventory of spectrometry equipment and illustrate the application of mass spectrometry of nucleic acids (DNA and RNA) for solving practical problems related to the analysis of human genomic DNA and clinically significant microorganisms of bacterial and viral natures. PMID:19537166

Il'ina, E N; Govorun, V M

2009-01-01

194

DRAWNA: a program for drawing schematic views of nucleic acids.  

PubMed

A program for drawing automatically exact and schematic views of nucleic acids is described. The program is written in C ANSI and uses the Silicon Graphics GL and Xirisw libraries within the X11/Motif environment. Through menus, the user can choose, specify, and manipulate in real time the three-dimensional views to be displayed. Drawing options include partitioning of structures into differently colored or shaped fragments, representation of backbones as flat or with conic-section ribbons, display of paired or free bases as rods, and display of surfaces as filled or outlined and stereo or depth-cued views. PMID:7529557

Massire, C; Gaspin, C; Westhof, E

1994-09-01

195

Generating non-overlapping displays of nucleic acid secondary structure.  

PubMed Central

A new algorithm is presented which permits the display of nucleic acid secondary structure by computer. This algorithm circumvents the problem of overlapping portions of the molecule which is inherent in some other drawing programs. The results from this algorithm may also be used as input to the drawing algorithm previously reported in this journal [1] to untangle most of a drawing. The algorithm also represents the molecule in a form which makes visual comparisons for similarity quite easy since it guarantees that comparable features will reside in the same relative position in the drawings when the drawings are normalized. PMID:6694904

Shapiro, B A; Maizel, J; Lipkin, L E; Currey, K; Whitney, C

1984-01-01

196

Nucleic acid modifications in bacterial pathogens - impact on pathogenesis, diagnosis, and therapy  

E-print Network

Nucleic acids are subject to extensive chemical modification by all organisms. These modifications display incredible structural diversity, and some are essential for survival. Intriguingly, several of these modifications ...

Russell, Brandon S. (Brandon Skylar)

2014-01-01

197

Frequent Detection of Viral Nucleic Acids in Heart Valve Tissue  

PubMed Central

Due to a paucity of published data concerning the prevalence of viral nucleic acid in homografts, we analyzed tissue from 30 donor hearts for the presence of viral genome sequences of enteroviruses, adenoviruses, human cytomegalovirus, and influenza virus using different PCR techniques. Viral DNA was amplified in 64 and 52% of the subvalvular myocardial tissue and non-coronary valve samples, respectively. These findings, compared with clinical history and histologic and serologic analysis, demonstrate the importance of viral safety measures in heart valve banking. PMID:15131218

Mantke, Oliver Donoso; Meyer, Rudolf; Prosch, Susanna; Niedrig, Matthias

2004-01-01

198

Antibodies specific for nucleic acids and applications in genomic detection and clinical diagnostics.  

PubMed

Detection of nucleic acids using antibodies is uncommon. This is in part because nucleic acids are poor immunogens and it is difficult to elicit antibodies having high affinity to each type of nucleic acid while lacking cross-reactivity to others. We describe the origins and applications of a variety of anti-nucleic acid antibodies, including ones reacting with modified nucleosides and nucleotides, single-stranded DNA, double-stranded DNA, RNA, DNA:RNA hybrids, locked-nucleic acids or peptide nucleic acid:nucleic acid hybrids. Carefully selected antibodies can be excellent reagents for detecting bacteria, viruses, small RNAs, microRNAs, R-loops, cancer cells, stem cells, apoptotic cells and so on. The detection may be sensitive, simple, rapid, specific, reproducible, quantitative and cost-effective. Current microarray and diagnostic methods that depend on cDNA or cRNA can be replaced by using antibody detection of nucleic acids. Therefore, development should be encouraged to explore new utilities and create a robust arsenal of new anti-nucleic acid antibodies. PMID:25014728

Hu, Zonglin; Leppla, Stephen H; Li, Baoguang; Elkins, Christopher A

2014-09-01

199

Nonenzymatic catalytic signal amplification for nucleic acid hybridization assays  

NASA Technical Reports Server (NTRS)

Devices, methods, and kits for amplifying the signal from hybridization reactions between nucleic acid probes and their cognate targets are presented. The devices provide partially-duplexed, immobilized probe complexes, spatially separate from and separately addressable from immobilized docking strands. Cognate target acts catalytically to transfer probe from the site of probe complex immobilization to the site of immobilized docking strand, generating a detectable signal. The methods and kits of the present invention may be used to identify the presence of cognate target in a fluid sample.

Fan, Wenhong (Inventor); Cassell, Alan M. (Inventor); Han, Jie (Inventor)

2006-01-01

200

Point of Attachment and Sequence of Immobilized Peptide-Acridine Conjugates Control Affinity for Nucleic Acids  

E-print Network

for Nucleic Acids Coby B. Carlson and Peter A. Beal* Department of Chemistry, UniVersity of Utah, 315 South of peptide-acridine conjugates (PACs) featuring a novel 9-anilinoacridine amino acid that we wish to screen if immobilization of a PAC affects binding to RNA targets. Similar compounds have been shown to bind nucleic acids

Beal, Peter A.

201

Design, preparation and application of nucleic acid delivery carriers.  

PubMed

Gene delivery vectors must deliver their cargoes into the cytosol or the nucleus, where DNA or siRNA functions in vivo. Therefore it is crucial for the rational design of the nucleic acid delivery carriers. Compared with viral vectors, non-viral vectors have overcome some fatal defections in gene therapy. Whereas the most important issue for the non-viral vectors is the low transfection efficiency, which hinders the progress of non-viral carriers. Sparked by the structures of the virus and understanding of the process of virus infection, various biomimic structures of non-viral carriers were designed and prepared to improve the transfection issues in vitro and in vivo. However, less impressive results are achieved. In this review, we will investigate the evolution of the virus-mimicking carriers of nucleic acids for gene therapy, especially in cancer therapy; explore and discuss the relationship between the structures, materials and functions of the carriers, to provide guidance for establishing safe and highly efficient non-viral carriers for gene therapy. PMID:24239630

Yang, Jun; Liu, Hongmei; Zhang, Xin

2014-01-01

202

Coarse-grained model of nucleic acid bases.  

PubMed

Atomistic simulations of nucleic acids are prohibitively expensive and, consequently, reduced models of these compounds are of great interest in the field. In this work, we propose a physics-based coarse-grained model of nucleic-acid bases in which each base is represented by several (3-5) interaction centers. van der Waals interactions are modeled by Lennard-Jones spheres with a 12-6 potential energy function. The charge distribution is modeled by a set of electric dipole moments located at the centers of the Lennard-Jones spheres. The method for computing the Lennard-Jones parameters, electric dipole moments (their magnitude and orientation) and positions of the interaction centers is described. Several models with different numbers of interaction centers were tested. The model with three-center cytosine, four-center guanine, four-center thymine, and five-center adenine satisfactorily reproduces the canonical Watson-Crick hydrogen bonding and stacking interaction energies of the all-atom AMBER model. The computation time with the coarse-grained model is reduced seven times compared with that of the all-atom model. PMID:20020472

Maciejczyk, Maciej; Spasic, Aleksandar; Liwo, Adam; Scheraga, Harold A

2010-06-01

203

UNAFold: software for nucleic acid folding and hybridization.  

PubMed

The UNAFold software package is an integrated collection of programs that simulate folding, hybridization, and melting pathways for one or two single-stranded nucleic acid sequences. The name is derived from "Unified Nucleic Acid Folding." Folding (secondary structure) prediction for single-stranded RNA or DNA combines free energy minimization, partition function calculations and stochastic sampling. For melting simulations, the package computes entire melting profiles, not just melting temperatures. UV absorbance at 260 nm, heat capacity change (C(p)), and mole fractions of different molecular species are computed as a function of temperature. The package installs and runs on all Unix and Linux platforms that we have looked at, including Mac OS X. Images of secondary structures, hybridizations, and dot plots may be computed using common formats. Similarly, a variety of melting profile plots is created when appropriate. These latter plots include experimental results if they are provided. The package is "command line" driven. Underlying compiled programs may be used individually, or in special combinations through the use of a variety of Perl scripts. Users are encouraged to create their own scripts to supplement what comes with the package. This evolving software is available for download at http://www.bioinfo.rpi.edu/applications/hybrid/download.php . PMID:18712296

Markham, Nicholas R; Zuker, Michael

2008-01-01

204

Nucleic acid sequence detection using multiplexed oligonucleotide PCR  

DOEpatents

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.

Nolan, John P. (Santa Fe, NM); White, P. Scott (Los Alamos, NM)

2006-12-26

205

Prebiotically plausible mechanisms increase compositional diversity of nucleic acid sequences  

PubMed Central

During the origin of life, the biological information of nucleic acid polymers must have increased to encode functional molecules (the RNA world). Ribozymes tend to be compositionally unbiased, as is the vast majority of possible sequence space. However, ribonucleotides vary greatly in synthetic yield, reactivity and degradation rate, and their non-enzymatic polymerization results in compositionally biased sequences. While natural selection could lead to complex sequences, molecules with some activity are required to begin this process. Was the emergence of compositionally diverse sequences a matter of chance, or could prebiotically plausible reactions counter chemical biases to increase the probability of finding a ribozyme? Our in silico simulations using a two-letter alphabet show that template-directed ligation and high concatenation rates counter compositional bias and shift the pool toward longer sequences, permitting greater exploration of sequence space and stable folding. We verified experimentally that unbiased DNA sequences are more efficient templates for ligation, thus increasing the compositional diversity of the pool. Our work suggests that prebiotically plausible chemical mechanisms of nucleic acid polymerization and ligation could predispose toward a diverse pool of longer, potentially structured molecules. Such mechanisms could have set the stage for the appearance of functional activity very early in the emergence of life. PMID:22319215

Derr, Julien; Manapat, Michael L.; Rajamani, Sudha; Leu, Kevin; Xulvi-Brunet, Ramon; Joseph, Isaac; Nowak, Martin A.; Chen, Irene A.

2012-01-01

206

Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corroded carbon steel  

Microsoft Academic Search

Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA\\/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high NA yield, quality, purity, representativeness of microbial community and processing time efficiency). The PowerSoil RNA Isolation Kit was

Florence Marty; Jean-François Ghiglione; Sandrine Païssé; Hervé Gueuné; Laurent Quillet; Mark C. M. van Loosdrecht; Gerard Muyzer

2012-01-01

207

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

PubMed Central

The objective of this study was to develop a novel peptide nucleic acid (PNA) probe for Stenotrophomonas maltophilia identification by fluorescence in situ hybridization (FISH). The probe was evaluated using 33 human and veterinary clinical S. maltophilia isolates and 45 reference strains representing common bacterial species in the respiratory tract. The probe displayed 100% sensitivity and 100% specificity on pure cultures and allowed detection in sputum from cystic fibrosis patients. The detection limit was 104 CFU/mL in spiked tracheal aspirate and bronchoalveolar lavage from healthy horses. Altogether the study shows that this species-specific PNA FISH probe facilitates rapid detection of S. maltophilia in biological specimens. PMID:25356348

Hansen, N; Rasmussen, A K I; Fiandaca, M J; Kragh, K N; Bjarnsholt, T; H?iby, N; Stender, H; Guardabassi, L

2014-01-01

208

Heat-labile alkaline phosphatase from Antarctic bacteria: Rapid 5? end-labeling of nucleic acids  

PubMed Central

A heat-labile alkaline phosphatase has been purified to near homogeneity from HK47, a bacterial strain isolated from Antarctic seawater. The active form of the enzyme has a molecular weight of 68,000 and is uniquely monomeric. The optimal temperature for the enzymatic activity is 25°C. Complete and irreversible thermal inactivation of the enzyme occurs in 10 min at 55°C. By using this heat-labile enzyme for dephosphorylation followed by a 10-min heat treatment, rapid end-labeling of nucleic acids by T4 polynucleotide kinase has been achieved. Images PMID:16593525

Kobori, Hiromi; Sullivan, Cornelius W.; Shizuya, Hiroaki

1984-01-01

209

Molecular cytogenetics by polymerase catalyzed amplification or in situ labelling of specific nucleic acid sequences  

SciTech Connect

The Polymerase Chain Reaction (PCR) can be performed on isolated cells or chromosomes and the product can be analyzed by DNA technology or by FISH to test metaphases. The authors have good experiences analyzing aberrant chromosomes by FACS sorting, PCR with degenerated primers and painting of test metaphases with the PCR product. They also utilize polymerases for PRimed IN Situ labelling (PRINS) of specific nucleic acid sequences. In PRINS oligonucleotides are hybridized to their target sequences and labeled nucleotides are incorporated at the site of hybridization with the oligonucleotide as primer. PRINS may eventually allow the study of individual genes, gene expression and even somatic mutations (in mRNA) in single cells.

Bolund, L.; Brandt, C.; Hindkjaer, J.; Koch, J.; Koelvraa, S.; Pedersen, S. (Univ. of Aarhus (Denmark))

1993-01-01

210

Compatible solute influence on nucleic acids: Many questions but few answers  

PubMed Central

Compatible solutes are small organic osmolytes including but not limited to sugars, polyols, amino acids, and their derivatives. They are compatible with cell metabolism even at molar concentrations. A variety of organisms synthesize or take up compatible solutes for adaptation to extreme environments. In addition to their protective action on whole cells, compatible solutes display significant effects on biomolecules in vitro. These include stabilization of native protein and nucleic acid structures. They are used as additives in polymerase chain reactions to increase product yield and specificity, but also in other nucleic acid and protein applications. Interactions of compatible solutes with nucleic acids and protein-nucleic acid complexes are much less understood than the corresponding interactions of compatible solutes with proteins. Although we may begin to understand solute/nucleic acid interactions there are only few answers to the many questions we have. I summarize here the current state of knowledge and discuss possible molecular mechanisms and thermodynamics. PMID:18522725

Kurz, Matthias

2008-01-01

211

Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes  

DOEpatents

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.

Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

2013-07-23

212

Gefitinib for non-small-cell lung cancer patients with epidermal growth factor receptor gene mutations screened by peptide nucleic acid-locked nucleic acid PCR clamp  

Microsoft Academic Search

This study was prospectively designed to evaluate a phase II study of gefitinib for non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations. Clinical samples were tested for EGFR mutations by peptide nucleic acid-locked nucleic acid PCR clamp, and patients having EGFR mutations were given gefitinib 250 mg daily as the second treatment after chemotherapy. Poor PS

A Sutani; Y Nagai; K Udagawa; Y Uchida; N Koyama; Y Murayama; T Tanaka; H Miyazawa; M Nagata; M Kanazawa; K Hagiwara; K Kobayashi

2006-01-01

213

Single-molecule characterization and engineering of the surfaces of nucleic acid sensors  

NASA Astrophysics Data System (ADS)

The advent of personalized medicine will require biosensors capable of reliably detecting small levels of disease biomarkers. In microarrays and sensors for nucleic acids, hybridization events between surface-tethered DNA probes and the nucleic acids of interest (targets) are transduced into a detectable signal. However, target-binding ultimately occurs as a result of molecular motions and interactions between the probe and target at the nanometer scale, and common characterization methods either lack the resolution to characterize the sensors at this scale or provide only limited information about their interactions with their nanoscale chemical environment. In this dissertation I argue that an impediment to the development of more reliable and practical biosensors is the lack of knowledge and control of the nanometer length-scale structure of biosensor surfaces, which has a profound impact on molecular recognition and reactions for detection. After reviewing the fundamental surface chemistry and structural motifs of biosensors in Chapter 1, in Chapter 2 I use electrochemical atomic force microscopy (EC-AFM) to characterize in situ a common class of model nucleic acid sensors---thiolated DNA attached to a gold electrode which has been passivated by an alkanethiol self-assembled monolayer---with single-molecule resolution. This level of detail allows me to observe both the conformations of individual probes and their spatial distribution at the nanoscale, then determine how these are affected by assembly conditions, probe structure, and interactions with co-adsorbates. I also determine how these nanoscale details affect the dynamic response of probes to electric fields, which have been commonly used in sensing schemes, and ultimately the ability of the surface-tethered probes to bind with target nucleic acids. In Chapter 3, I demonstrate and optimize the nanoscale patterning of individual DNA molecules into isolated, chemically well-defined niches on the surface, and the use of these patterned probes as a single-molecule `nano-array' able to bind with target nucleic acids. Additionally, an outstanding issue is the expense of the high-quality substrates used in these studies. In Chapter 4, I discuss the development of single-crystal gold micro-plates with controllable surface chemistries as high-quality substrates for biotechnological platforms at a fraction of the cost.

Josephs, Eric Alan

214

Honey, I Shrunk the DNA: DNA Length as a Probe for Nucleic-Acid Enzyme Activity  

E-print Network

Honey, I Shrunk the DNA: DNA Length as a Probe for Nucleic-Acid Enzyme Activity Antoine M. van movies'' of enzymes at the single-molecule level, and thus remove averaging over large numbers to manipulate individual DNA molecules17 have allowed a large number of nucleic-acid enzymes to be charac

215

Cells labeled with multiple fluorophores bound to a nucleic acid carrier  

SciTech Connect

In passing labeled cells through a cell sorter, the improvement which comprises employing a labeled cell comprising a cell, an antibody specific to and bound to such cell, a nucleic acid fragment joined to the antibody, and a plurality of labels on the nucleic acid fragment. Because of the presence of multiple labels, the sensitivity of the separation of labeled cells in increased.

Dattagupta, N.; Kamarch, M.E.

1989-04-25

216

Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters  

Microsoft Academic Search

Nucleic acid-based therapies hold great promise for treatment of skin disorders if delivery challenges can be overcome. To investigate one mechanism of nucleic acid delivery to keratinocytes, a fixed mass of expression plasmid was intradermally injected into mouse footpads in different volumes, and reporter expression was monitored by intravital imaging or skin sectioning. Reporter gene expression increased with higher delivery

E González-González; H Ra; R Spitler; R P Hickerson; C H Contag; R L Kaspar

2010-01-01

217

APPLICATION OF THE RATE OF NUCLEIC ACID SYNTHESIS TO THE STUDY OF MICROBIAL GROWTH  

E-print Network

, extrapolated from DNA synthesis measurements, and 14C02 primary production on a transect of the equatorialAPPLICATION OF THE RATE OF NUCLEIC ACID SYNTHESIS TO THE STUDY OF MICROBIAL GROWTH AND PRODUCTION. Stroup Tom Humphreys #12;ABSTRACT The rate of nucleic acid synthesis was used as a measure of growth

Luther, Douglas S.

218

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

PubMed Central

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. PMID:24452173

Kalina, Warren Vincent; Douglas, Christina Elizabeth; Coyne, Susan Rajnik

2014-01-01

219

Real-time investigation of nucleic acids phosphorylation process using molecular beacons  

Microsoft Academic Search

Phosphorylation of nucleic acids is an indispensable process to repair strand interruption of nucleic acids. We have studied the process of phosphoryla- tion using molecular beacon (MB) DNA probes in real-time and with high selectivity. The MB employed in this method is devised to sense the product of a 'phosphorylation-ligation' coupled enzyme reaction. Compared with the current assays, this novel

Zhiwen Tang; Kemin Wang; Weihong Tan; Changbei Ma; Jun Li; Lingfeng Liu; Qiuping Guo; Xiangxian Meng

2005-01-01

220

Helicases and NTP-Driven Nucleic Acid Machines: Structure, Function and Roles in Human Disease  

E-print Network

EDITORIAL Helicases and NTP-Driven Nucleic Acid Machines: Structure, Function and Roles in Human the discovery of the structure of DNA we have known that nucleic acids can exist in a complementary, base of different machines that use NTP to perform their work, either to unwind the double-stranded molecule

Kowalczykowski, Stephen C.

221

Enzymatic synthesis of DNA on glycerol nucleic acid templates without stable duplex formation between  

E-print Network

Enzymatic synthesis of DNA on glycerol nucleic acid templates without stable duplex formation for review May 8, 2007) Glycerol nucleic acid (GNA) is an interesting alternative base- pairing system based on an acyclic, glycerol-phosphate backbone repeat unit. The question of whether DNA polymerases can cata- lyze

Heller, Eric

222

Evolution of functional nucleic acids in the presence of nonheritable backbone heterogeneity  

E-print Network

Evolution of functional nucleic acids in the presence of nonheritable backbone heterogeneity Simon that the early evolution of life was dominated by RNA, which can both transfer information from generation of heterogeneous nucleic acid molecules could evolve reproducible function. For such evolution to be possible

Heller, Eric

223

Versatile DNAzyme-Based Amplified Biosensing Platforms for Nucleic Acid, Protein, and Enzyme Activity Detection  

E-print Network

and concentrations of certain biomolecules (e.g., nucleic acids and proteins) inside a human body can reflect of the above-mentioned diseases, the concen- trations of their biomarkers in human body are usually very lowVersatile DNAzyme-Based Amplified Biosensing Platforms for Nucleic Acid, Protein, and Enzyme

Tan, Weihong

224

A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands  

Microsoft Academic Search

The mechanisms underlying a new hybridocytochemical method, which is based on mercurated nucleic acid probes and their binding to sulfhydryl-hapten ligands, have been studied. Furthermore we developed a simple procedure for the preparation of mercurated probes at a microgram scale. Nucleic acids immobilized on Sephadex beads have been immunochemically detected after hybridization with mercurated probes and binding of the sulfhydryl-hapten

A. H. N. Hopman; J. Wiegant; P. Duijn

1986-01-01

225

Integration of On-Chip Isotachophoresis and Functionalized Hydrogels for Enhanced-Sensitivity Nucleic Acid Detection  

E-print Network

introduce an on-chip electrokinetic assay to perform high-sensitivity nucleic acid (NA) detection-Sensitivity Nucleic Acid Detection Giancarlo Garcia-Schwarz and Juan G. Santiago* Department of Mechanical Engineering less than 100 fg of microRNA, and high selectivity for mature microRNA sequences, all within a 10 min

Santiago, Juan G.

226

Information transfer from DNA to peptide nucleic acids by template- directed syntheses  

Microsoft Academic Search

Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2

G. Schmidt; Leif Christensen; Peter E. Nielsen; Leslie E. Orgel

1997-01-01

227

Nucleic acid probes are molecules that complement or hybridize to a  

E-print Network

Nucleic acid probes are molecules that complement or hybridize to a specific mRNA or DNA sequence of rapid methods of nucleic acid sequencing and measurement made DNA and RNA the center of atten- tion computational methods of protein identification since analysis can involve thousands of peptide mass spectra

Levin, Judith G.

228

Telomerase inhibition by peptide nucleic acids reverses `immortality' of transformed human cells  

Microsoft Academic Search

Telomerase activity, the ability to add telomeric repeats to the ends of chromosomes, has been detected in most immortal cell lines including tumor cells, but is low or absent in most diploid, mortal cells such as those of somatic tissues. Peptide nucleic acids (PNAs), analogs of DNA or RNA which bind to complementary nucleic acids with very high affinity, were

Masood A Shammas; Carla G Simmons; David R Corey; Robert J Shmookler Reis; RJS Reis

1999-01-01

229

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

PubMed

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

Chikov, V M; Maksimova, E V

1989-01-01

230

Circulating and stool nucleic acid analysis for colorectal cancer diagnosis  

PubMed Central

In recent years, the need to identify molecular markers characterized by high sensitivity and specificity in detecting and monitoring early and colorectal cancer lesions has increased. Up to now, none of the markers or panels of markers analyzed have met the rigorous standards required of a screening program. The important discovery of circulating nucleic acids in biological fluids has aroused intense scientific interest because of their usefulness in malignant and non malignant diseases. Over time, their yield and stability have been identified and compared with other “standard” biomarkers. The analysis of circulating DNA from blood and stool is a relatively simple and non-invasive procedure, representing a very attractive marker to detect genetic and epigenetic mutations and to monitor disease progression. A correlation between blood and stool biomarkers could also help to enhance currently available diagnostic approaches. However, various processing and analytic problems need to be resolved before such an approach can be applied in clinical practice. PMID:24574768

De Maio, Giulia; Rengucci, Claudia; Zoli, Wainer; Calistri, Daniele

2014-01-01

231

Nucleic acids--genes, drugs, molecular lego and more.  

PubMed

Chemically modified nucleic acids find widespread use as tools in research, as diagnostic reagents and even as pharmaceutical compounds. On the background of antisense research and development, the synthesis and evaluation of modified oligonucleotides was intensively pursued in the early to mid nineties in corporate research of former Ciba. Most of these efforts concentrated on the development of sugar and/or backbone-modified derivatives for pharmaceutical applications. Additionally, oligonucleotide metal conjugates were investigated with the goal to develop artificial ribonucleases. Since the turn of the millennium also the potential of non-nucleosidic and non-hydrogen bonding building blocks has increasingly been recognized. Such derivatives possess unique properties that may have an impact in the fields of materials and genetic research. In this brief account, we take a personal look back on some past as well as some recent results. PMID:21137677

Häner, Robert

2010-01-01

232

Spherical Nucleic Acids: A New Form of DNA  

NASA Astrophysics Data System (ADS)

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 subsequently dissolved oxidatively with KCN or Iodine. The reaction pathway is analyzed through characterization of the reaction progression and resulting products, and a mechanistic pathway is proposed. This is the first report of a gold nanoparticle catalyzed reaction involving the conversion of propargyl ethers to terminal alcohols, which can subsequently cross-link if densely arranged on a gold nanoparticle surface. Importantly, these structures can be synthesized using gold nanoparticles of a range of sizes, thereby providing control over the size and properties of the resulting crosslinked particle. Chapter four returns to the topic of SNAs and builds upon the chemistry of chapter three culminating in the synthesis of cross-linked hollow SNA nanoparticles. These structures are formed by the cross-linking of synthetically modified alkyne-bearing oligonucleotides through the pathway described in chapter three. When the gold core is dissolved, the resulting hollow SNAs exhibit nearly identical binding, nuclease resistance, cellular uptake, and gene regulation properties of SNA-gold nanoparticle conjugates. Indeed, this chapter demonstrates that the unique properties of SNA-nanoparticle conjugates are core-independent and stem solely from the dense ensemble of oligonucleotides arranged on their surfaces. The fifth chapter further asserts the synthetic achievements made in chapter four by showing how hollow SNAs can be substituted for SNA-gold nanoparticles in the context of DNA-programmable assembly. In this case, they can be used as building blocks within binary synthetic schemes to synthesize unique nanoparticle superlattices. It bolsters the design rules of DNA-programmable assembly by showing that the predicted structures form based on the behavior of SNA hybridization, and are universal for any SNA-functionalized nanoparticle.

Cutler, Joshua Isaac

233

Nucleic Acid Encoding A Lectin-Derived Progenitor Cell Preservation Factor  

DOEpatents

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.

Colucci, M. Gabriella (Dugenta, IT); Chrispeels, Maarten J. (La Jolla, CA); Moore, Jeffrey G. (New York, NY)

2001-10-30

234

Electrostatics of nucleic acid folding under conformational constraint  

PubMed Central

RNA folding is enabled by interactions between the nucleic acid and its ion atmosphere, the mobile sheath of aqueous ions that surrounds and stabilizes it. Understanding the ion atmosphere requires the interplay of experiment and theory. However, even an apparently simple experiment to probe the ion atmosphere—measuring the dependence of DNA duplex stability upon ion concentration and identity—suffers from substantial complexity, because the unfolded ensemble contains many conformational states that are difficult to treat accurately with theory. To minimize this limitation, we measured the unfolding equilibrium of a DNA hairpin using a single-molecule optical trapping assay, in which the unfolded state is constrained to a limited set of elongated conformations. The unfolding free energy increased linearly with the logarithm of monovalent cation concentration for several cations, such that smaller cations tended to favor the folded state. Mg2+ stabilized the hairpin much more effectively at low concentrations than did any of the monovalent cations. Poisson-Boltzmann theory captured trends in hairpin stability measured for the monovalent cation titrations with reasonable accuracy, but failed to do so for the Mg2+ titrations. This finding is consistent with previous work suggesting that Poisson-Boltzmann and other mean-field theories fail for higher valency cations where ionion correlation effects may become significant. The high-resolution data herein, because of the straightforward nature of both the folded and unfolded states, should serve as benchmarks for the development of more accurate electrostatic theories that will be needed for a more quantitative and predictive understanding of nucleic acid folding. PMID:22369617

Anthony, Peter C.; Sim, Adelene Y.L.; Chu, Vincent B.; Doniach, Sebastian; Block, Steven M.; Herschlag, Daniel

2012-01-01

235

Polysaccharide-free nucleic acids and proteins of Abelmoschus esculentus for versatile molecular studies.  

PubMed

Abelmoschus esculentus (okra) is one of the polysaccharide rich crop plants. The polysaccharides interfere with nucleic acids and protein isolation thereby affecting the downstream molecular analysis. So, to understand the molecular systematics of okra, high quality DNA, RNA and proteins are essential. In this study we present a method for extracting genomic DNA, RNA and proteins from polysaccharide rich okra tissues. The conventional extraction procedures were integrated with purification treatments with pectinase, RNase and proteinase K, which improved the quality and quantity of DNA as well. Using SDS, additional washes with CIA and NaCl precipitation improved the RNA isolation both quantitatively and qualitatively. Finally, ammonium acetate mediated protein precipitation and re-solubilization increased the quality of total protein extracts from the okra leaves. All of the methods above not only eliminated the impurities but also improved the quality and quantity of nucleic acids and proteins. Further, we subjected these samples to versatile downstream molecular analyses such as restriction endonuclease digestion, RAPD, Southern, reverse transcription-PCR and Western analysis and were proved to be successful. PMID:23113348

Manoj-Kumar, A; Reddy, K N; Manjulatha, M; Blanco, L

2012-01-01

236

Helicases and nucleic acid translocases are a diverse group of motor proteins that function in nearly all  

E-print Network

single strands. Nucleic acid translocases, not all of which are helicases, use NTP to move with biased motifs are present in a wide range of NTP-dependent nucleic acid enzymes24 , many of whichHelicases and nucleic acid translocases are a diverse group of motor proteins that function

Lohman, Timothy M.

237

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

Microsoft Academic Search

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

Darren J. Carter; R. Bruce Cary

2007-01-01

238

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

PubMed

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 for therapeutic nucleic acid delivery strategies. PMID:20830348

Hansen, Pernille Lund; Blaich, Stephanie; End, Caroline; Schmidt, Steffen; Moeller, Jesper B; Holmskov, Uffe; Mollenhauer, Jan

2011-01-01

239

Nucleic acids and endosomal pattern recognition: how to tell friend from foe?  

PubMed Central

The innate immune system has evolved endosomal and cytoplasmic receptors for the detection of viral nucleic acids as sensors for virus infection. Some of these pattern recognition receptors (PRR) detect features of viral nucleic acids that are not found in the host such as long stretches of double-stranded RNA (dsRNA) and uncapped single-stranded RNA (ssRNA) in case of Toll-like receptor (TLR) 3 and RIG-I, respectively. In contrast, TLR7/8 and TLR9 are unable to distinguish between viral and self-nucleic acids on the grounds of distinct molecular patterns. The ability of these endosomal TLR to act as PRR for viral nucleic acids seems to rely solely on the mode of access to the endolysosomal compartment in which recognition takes place. The current dogma states that self-nucleic acids do not enter the TLR-sensing compartment under normal physiological conditions. However, it is still poorly understood how dendritic cells (DC) evade activation by self-nucleic acids, in particular with regard to specific DC subsets, which are specialized in taking up material from dying cells for cross-presentation of cell-associated antigens. In this review we discuss the current understanding of how the immune system distinguishes between foreign and self-nucleic acids and point out some of the key aspects that still require further research and clarification. PMID:23908972

Brencicova, Eva; Diebold, Sandra S.

2013-01-01

240

Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins  

NASA Astrophysics Data System (ADS)

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.

Williams, Mark

2010-03-01

241

Detection of Human Enteric Viruses in Oysters by In Vivo and In Vitro Amplification of Nucleic Acids  

Microsoft Academic Search

ThisstudydescribesthedetectionofenterovirusesandhepatitisAvirusin31naturallycontaminatedoyster specimens by nucleic acid amplification and oligonucleotide probing. Viruses were extracted by adsorption- elution-precipitation from 50-g oyster samples harvested from an area receiving sewage effluent discharge. Ninety percent of each extract was inoculated into primate kidney cell cultures for virus isolation and infectivity assay. Viruses in the remaining 10% of oyster extract that was not inoculated into cell cultures

HYENMI CHUNG; LEE-ANN JAYKUS; ANDMARK D. SOBSEY

1996-01-01

242

Incorporation of Glycine into Protein and Nucleic Acid Fractions of Nuclei of Liver and Hepatoma  

Microsoft Academic Search

SUMMARY Proteins and nucleic acids of nuclei of liver and hepatoma in the rat \\\\vere separated into three fractions: Fraction I, soluble in 0.05 Msodium citrate; Fraction II, extracted with 1 M NaCl; and Fraction III, the residual fraction. Histones, acid-insoluble pro teins, RNA, and DNA were found in each fraction. Specific activities of the protein and nucleic acid fractions

DAVID J. HOLBROOK; J. LOGAN; MOORE IRVIN

243

Synthesis of phosphoramidites of isoGNA, an isomer of glycerol nucleic acid  

PubMed Central

Summary IsoGNA, an isomer of glycerol nucleic acid GNA, is a flexible (acyclic) nucleic acid with bases directly attached to its linear backbone. IsoGNA exhibits (limited) base-pairing properties which are unique compared to other known flexible nucleic acids. Herein, we report on the details of the preparation of isoGNA phosphoramidites and an alternative route for the synthesis of the adenine derivative. The synthetic improvements described here enable an easy access to isoGNA and allows for the further exploration of this structural unit in oligonucleotide chemistry thereby spurring investigations of its usefulness and applicability.

Kim, Keunsoo; Punna, Venkateshwarlu; Karri, Phaneendrasai

2014-01-01

244

Carbon composite micro- and nano-tubes-based electrodes for detection of nucleic acids  

PubMed Central

The first aim of this study was to fabricate vertically aligned multiwalled carbon nanotubes (MWCNTs). MWCNTs were successfully prepared by using plasma enhanced chemical vapour deposition. Further, three carbon composite electrodes with different content of carbon particles with various shapes and sizes were prepared and tested on measuring of nucleic acids. The dependences of adenine peak height on the concentration of nucleic acid sample were measured. Carbon composite electrode prepared from a mixture of glassy and spherical carbon powder and MWCNTs had the highest sensitivity to nucleic acids. Other interesting result is the fact that we were able to distinguish signals for all bases using this electrode. PMID:21711910

2011-01-01

245

Synthesis and properties of carbohydrate-phosphate backbone-modified oligonucleotide analogues and nucleic acid mimetics  

NASA Astrophysics Data System (ADS)

Advances in the synthesis of oligo(deoxy)ribonucleotide analogues and nucleic acid mimetics made in the last decade are summarized. Attention is focused on new methods for the synthesis of derivatives with a modified ribose-phosphate backbone (phosphorothioate, boranophosphate, and nucleoside phosphonate derivatives) and derivatives devoid of the phosphate group. Among nucleic acid mimetics, conformationally restricted modified peptide nucleic acids, including those bearing a negative or positive charge, and morpholino oligomers are considered. Advantages and drawbacks of the main types of analogues as regards the complexity of the synthesis and the possibility of their application as antisense agents or reagents for hybridization analysis are compared.

Abramova, Tatyana V.; Silnikov, Vladimir N.

2011-05-01

246

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

NASA Technical Reports Server (NTRS)

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.

Setlow, Richard B.

2002-01-01

247

Synthesis and characterization of peptide nucleic acid platinum nanoclusters  

NASA Astrophysics Data System (ADS)

Peptide nucleic acid (PNA) is an analogue of deoxyribonucleic acid (DNA) and possesses a neutral backbone that affords stronger hybridization, greater stability and higher specificity in base pairing. However, it has not been explored as much as DNA in self-assembling functional nanostructures or nanoelectronic devices. We report here for the first time the metallization of PNA with platinum (Pt) nanoparticles via chemical binding, reduction and deposition. Pt ions from a precursor salt solution are allowed to bind over the PNA fragments followed by a reduction and then growth into metal nanoparticles. PNA-Pt complexes form chains several hundred nanometres in length and by varying the duration of chemical reduction step, the dimension of the Pt nanoparticles can be controlled. The structural features and chemical composition of PNA-Pt nanoparticles have been characterized via scanning electron microscopy, transmission electron microscopy and Fourier transform-infrared spectroscopy. These results are also supported by modelling and analysis of the nature of high-lying molecular orbitals on PNA using density functional theory (DFT) method.

Wang, Xu; Pandey, Rajeev R.; Singh, Krishna V.; Senthil Andavan, G. T.; Tsai, Chunglin; Lake, Roger; Ozkan, Mihrimah; Ozkan, Cengiz S.

2006-03-01

248

Nonisotopic detection methods for strand displacement assays of nucleic acids.  

PubMed

Using the enzymes terminal deoxyribonucleotidyltransferase (EC 2.7.7.31) and polynucleotide phosphorylase (EC 2.7.7.8), we constructed polyriboadenylic acid tracts, approximately 8000 AMP residues long, attached to the 3'-terminus of a synthetic deoxynucleotide. The polyadenylated DNA, termed the "signal strand", was used in a displacement-type nucleic acid probe assay (see pp 1631-6, this issue). A probe-signal strand complex was made by hybridizing the signal strand to a deoxycytidylate-terminal probe DNA. The probe-signal strand complex was immobilized on an oligo (dG)-cellulose support and subsequently displaced from the immobilized hybrid complex with various amounts of analyte DNA. After the displacement procedure, the polyadenylate tracts were converted to ATP by the combined action of polynucleotide phosphorylase and pyruvate kinase. ATP was quantified by a bioluminescence assay with luciferase from Photinus pyralis. Displacement events were also quantified with biotinylated signal strand bound to avidin-conjugated horseradish peroxidase. Such enzyme-amplified assays offer considerable versatility: they may be coupled to a variety of detection systems including colorimetry, fluorimetry, and luminometry. PMID:2427259

Vary, C P; McMahon, F J; Barbone, F P; Diamond, S E

1986-09-01

249

Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein  

PubMed Central

Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins. PMID:24339931

Freire, Joao Miguel; Veiga, Ana Salome; Conceicao, Thais M.; Kowalczyk, Wioleta; Mohana-Borges, Ronaldo; Andreu, David; Santos, Nuno C.; Da Poian, Andrea T.; Castanho, Miguel A. R. B.

2013-01-01

250

Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems?  

Microsoft Academic Search

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best

PHILIPPE LEBARON; PIERRE SERVAIS; HELENE AGOGUE; CLAUDE COURTIES; FABIEN JOUX

2001-01-01

251

Method for producing labeled single-stranded nucleic acid probes  

DOEpatents

Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

Dunn, John J. (Bellport, NY); Quesada, Mark A. (Middle Island, NY); Randesi, Matthew (Upton, NY)

1999-10-19

252

Plants having modified response to ethylene by transformation with an ETR nucleic acid  

DOEpatents

The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.

Meyerowitz, Elliott M. (Pasadena, CA); Chang, Caren (Pasadena, CA); Bleecker, Anthony B. (Madison, WI)

2001-01-01

253

Thermoplastic Microfluidic Device for On-Chip Purification of Nucleic Acids for Disposable  

E-print Network

Thermoplastic Microfluidic Device for On-Chip Purification of Nucleic Acids for Disposable of disposable, point-of-care microfluidic chips. The most promising class of materials is thermoplastic polymers

254

Two-dimensional infrared spectroscopy of nucleic acids : application to tautomerism and DNA aptamer unfolding dynamics  

E-print Network

The structural dynamics of nucleic acids are intimately related to their biological functions; however, our ability to study these molecular dynamics has been largely impeded by the lack of techniques that possess both ...

Peng, Chunte Sam

2014-01-01

255

Development of polymer and lipid materials for enhanced delivery of nucleic acids and proteins  

E-print Network

The development of synthetic vectors enabling efficient intracellular delivery of macromolecular therapeutics such as nucleic acids and proteins could potentially catalyze the clinical translation of many gene and protein-based ...

Eltoukhy, Ahmed Atef

2013-01-01

256

To Build a Virus on a Nucleic Acid Substrate  

PubMed Central

Many viruses package their genomes concomitant with assembly. Here, we show that this reaction can be described by three coefficients: association of capsid protein (CP) to nucleic acid (NA), KNA; CP-CP interaction, ?; and ?, proportional to the work required to package NA. The value of ? can vary as NA is packaged. A phase diagram of average ln? versus ln? identifies conditions where assembly is likely to fail or succeed. NA morphology can favor (ln? > 0) or impede (ln? < 0) assembly. As ln? becomes larger, capsids become more stable and assembly becomes more cooperative. Where (ln? + ln?) < 0, the CP is unable to contain the NA, so that assembly results in aberrant particles. This phase diagram is consistent with quantitative studies of cowpea chlorotic mottle virus, hepatitis B virus, and simian virus 40 assembling on ssRNA and dsDNA substrates. Thus, the formalism we develop is suitable for describing and predicting behavior of experimental studies of CP assembly on NA. PMID:23561536

Zlotnick, Adam; Porterfield, J. Zachary; Wang, Joseph Che-Yen

2013-01-01

257

Understanding barriers to efficient nucleic acid delivery with bioresponsive block copolymers  

E-print Network

The delivery of nucleic acids has the potential to revolutionize medicine by allowing previously untreatable diseases to be clinically addressed. Viral delivery systems have been held back by immunogenicity and toxicity ...

Bonner, Daniel Kenneth

2012-01-01

258

G-quadruplex nucleic acids and human disease  

PubMed Central

Alternate DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by sequences that are widely distributed throughout the human genome. G-quadruplex secondary structures, formed by the stacking of planar quartets composed of four guanines that interact by Hoogsteen hydrogen bonding, can affect cellular DNA replication and transcription, and influence genomic stability. The unique metabolism of G-rich chromosomal regions that potentially form quadruplexes may influence a number of biological processes including immunoglobulin gene rearrangements, promoter activation and telomere maintenance. A number of human diseases are characterized by telomere defects, and it is proposed that G-quadruplex structures which form at telomere ends play an important role in telomere stability. Evidence from cellular studies and model organisms suggests that diseases with known defects in G4 DNA helicases are likely to be perturbed in telomere maintenance and cellular DNA replication. In this minireview, we discuss the connections of G-quadruplex nucleic acids to human genetic diseases and cancer based on the recent literature. PMID:20670277

Wu, Yuliang; Brosh, Robert M.

2010-01-01

259

Monitoring Gene Expression In Vivo with Nucleic Acid Molecular Switches  

SciTech Connect

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.

David C. Ward; Patricia Bray-Ward

2005-01-26

260

Improved nucleic acid descriptors for siRNA efficacy prediction  

PubMed Central

Although considerable progress has been made recently in understanding how gene silencing is mediated by the RNAi pathway, the rational design of effective sequences is still a challenging task. In this article, we demonstrate that including three-dimensional descriptors improved the discrimination between active and inactive small interfering RNAs (siRNAs) in a statistical model. Five descriptor types were used: (i) nucleotide position along the siRNA sequence, (ii) nucleotide composition in terms of presence/absence of specific combinations of di- and trinucleotides, (iii) nucleotide interactions by means of a modified auto- and cross-covariance function, (iv) nucleotide thermodynamic stability derived by the nearest neighbor model representation and (v) nucleic acid structure flexibility. The duplex flexibility descriptors are derived from extended molecular dynamics simulations, which are able to describe the sequence-dependent elastic properties of RNA duplexes, even for non-standard oligonucleotides. The matrix of descriptors was analysed using three statistical packages in R (partial least squares, random forest, and support vector machine), and the most predictive model was implemented in a modeling tool we have made publicly available through SourceForge. Our implementation of new RNA descriptors coupled with appropriate statistical algorithms resulted in improved model performance for the selection of siRNA candidates when compared with publicly available siRNA prediction tools and previously published test sets. Additional validation studies based on in-house RNA interference projects confirmed the robustness of the scoring procedure in prospective studies. PMID:23241392

Sciabola, Simone; Cao, Qing; Orozco, Modesto; Faustino, Ignacio; Stanton, Robert V.

2013-01-01

261

Phospholipid conjugate for intracellular delivery of peptide nucleic acids  

PubMed Central

Peptide nucleic acids (PNAs) have a number of attractive features that have made them an ideal choice for antisense and antigene-based tools, probes and drugs, but their poor membrane permeability has limited their application as therapeutic or diagnostic agents. Herein we report a general method for the synthesis of phospholipid-PNAs (LP-PNAs), and compare the effect of non-cleavable lipids and bioreductively cleavable lipids (L and LSS) and phospholipid (LP) on the splice-correcting bioactivity of a PNA bearing the cell penetrating Arg9 group (PNA-R9). While the three constructs show similar and increasing bioactivity at 1–3 ?M, the activity of LP-PNA-R9 continues to increase from 4–6 ?M while the activity of L-PNA-R9 remains constant and LSS-PNA-R9 decreases rapidly in parallel with their relative cytotoxicity. The activity of both LP-PNA-R9 and L-PNA-R9 were found to dramatically increase with chloroquine, as expected for an endocytotic entry mechanism. Both constructs were also found to have CMC values of 1.0 and 4.5 ?M in 150 mM NaCl, pH 7 water, suggesting that micelle formation may play a hitherto unrecognized role in modulating toxicity and/or facilitating endocytosis. PMID:19678628

Shen, Gang; Fang, Huafeng; Song, Yinyin; Bielska, Agata A.; Wang, Zhenghui; Taylor, John-Stephen A.

2009-01-01

262

Ligation with nucleic acid sequence-based amplification.  

PubMed

This work presents a novel method for detecting nucleic acid targets using a ligation step along with an isothermal, exponential amplification step. We use an engineered ssDNA with two variable regions on the ends, allowing us to design the probe for optimal reaction kinetics and primer binding. This two-part probe is ligated by T4 DNA Ligase only when both parts bind adjacently to the target. The assay demonstrates that the expected 72-nt RNA product appears only when the synthetic target, T4 ligase, and both probe fragments are present during the ligation step. An extraneous 38-nt RNA product also appears due to linear amplification of unligated probe (P3), but its presence does not cause a false-positive result. In addition, 40 mmol/L KCl in the final amplification mix was found to be optimal. It was also found that increasing P5 in excess of P3 helped with ligation and reduced the extraneous 38-nt RNA product. The assay was also tested with a single nucleotide polymorphism target, changing one base at the ligation site. The assay was able to yield a negative signal despite only a single-base change. Finally, using P3 and P5 with longer binding sites results in increased overall sensitivity of the reaction, showing that increasing ligation efficiency can improve the assay overall. We believe that this method can be used effectively for a number of diagnostic assays. PMID:22449695

Ong, Carmichael; Tai, Warren; Sarma, Aartik; Opal, Steven M; Artenstein, Andrew W; Tripathi, Anubhav

2012-01-01

263

Activatable cell penetrating peptide-peptide nucleic acid conjugate via reduction of azobenzene PEG chains.  

PubMed

The use of stimuli-responsive bioactive molecules is an attractive strategy to circumvent selectivity issues in vivo. Here, we report an activatable cell penetrating peptide (CPP) strategy ultimately aimed at delivering nucleic acid drugs to the colon mucosa using bacterial azoreductase as the local reconversion trigger. Through screening of a panel of CPPs, we identified a sequence (M918) capable of carrying a nucleic acid analogue payload. A modified M918 peptide conjugated to a peptide nucleic acid (PNA) was shown to silence luciferase in colon adenocarcinoma cells (HT-29-luc). Reversible functionalization of the conjugate's lysine residues via an azobenzene self-immolative linkage abolished transfection activity, and the free CPP-PNA was recovered after reduction of the azobenzene bond. This activatable CPP conjugate platform could find applications in the selective delivery of nucleic acid drugs to the colon mucosa, opening therapeutic avenues in colon diseases. PMID:25185512

Lee, Soo Hyeon; Moroz, Elena; Castagner, Bastien; Leroux, Jean-Christophe

2014-09-17

264

A nucleic acid-based medication for allergic skin diseases.  

PubMed

Among allergic skin diseases, atopic dermatitis is the most difficult to cure. In the majority of patients, atopic dermatitis can be easily controlled by treatment based on three therapeutic approaches: avoidance of precipitating factors, skin care, and medication. In some adult patients, however, severe atopic dermatitis is refractory to treatment, and no fundamental effective treatment modality has yet been established for such cases. Chronic contact dermatitis without an identified causative hapten is also considered an allergic skin disease that is difficult to cure. Topical nucleic acid-based medications are currently being applied clinically, and an ointment containing nuclear factor-?B decoy oligodeoxynucleotides (hereafter referred to as Decoy) has reached clinical trials. In addition, synthetic double-stranded DNA with high affinity for signal transducers and activators of transcription 6 (STAT6) introduced in vivo as a decoy cis element to bind the transcriptional factor and block the activated gene that contributes to the onset and progression of atopic dermatitis functions as an effective therapeutic agent. We also introduce another STAT1 decoy treatment, cytosine-phosphate-guanine-ODN or STAT6 small interfering RNA therapy, for allergic skin diseases. PMID:24726501

Yokozeki, Hiroo

2014-08-01

265

Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids  

Microsoft Academic Search

Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination\\u000a with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms\\u000a (SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids\\u000a and SNPs in PNA\\/DNA and DNA\\/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles

Kagan Kerman; Masato Saito; Eiichi Tamiya

2008-01-01

266

Inhibition of the bovine papillomavirus E2 protein activity by peptide nucleic acid  

Microsoft Academic Search

The bovine papillomavirus type-1 E2 protein is the master regulator of the papillomavirus transcription and replication, the activity of which is regulated through sequence-specific DNA binding. Peptide nucleic acid (PNA) is a nucleic acid analogue, which associates with high affinity to complementary DNA, RNA or PNA, yielding in formation of stable complexes. The potential use of PNA as a sequence-specific

Reet Kurg; Ülo Langel; Mart Ustav

2000-01-01

267

Affinity Purification of DNA and RNA from Environmental Samples with Peptide Nucleic Acid Clamps  

Microsoft Academic Search

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sed- iment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO4, 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate (SDS)) a PNA clamp recovered significantly more

DARRELL P. CHANDLER; JENNIE R. STULTS; SHARON CEBULA; BEATRICE L. SCHUCK; DEREK W. WEAVER; KEVIN K. ANDERSON; MICHAEL EGHOLM; FRED J. BROCKMAN

2000-01-01

268

Plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases  

Microsoft Academic Search

Plasmacytoid dendritic cells (pDCs) are important mediators of antiviral immunity through their ability to produce large amounts of type I interferons (IFNs) on viral infection. This function of pDCs is linked to their expression of Toll-like receptor 7 (TLR7) and TLR9, which sense viral nucleic acids within the early endosomes. Exclusion of self nucleic acids from TLR-containing early endosomes normally

Michel Gilliet; Wei Cao; Yong-Jun Liu

2008-01-01

269

5'to 3' nucleic acid synthesis using 3'-photoremovable protecting group  

DOEpatents

The present invention relates, in general, to a method of synthesizing a nucleic acid, and, in particular, to a method of effecting 5' to 3' nucleic acid synthesis. The method can be used to prepare arrays of oligomers bound to a support via their 5' end. The invention also relates to a method of effecting mutation analysis using such arrays. The invention further relates to compounds and compositions suitable for use in such methods.

Pirrung, Michael C. (Houston, TX); Shuey, Steven W. (Durham, NC); Bradley, Jean-Claude (Durham, NC)

1999-01-01

270

Trimethylsilyl derivatization of nucleic acid anions in the gas phase  

NASA Astrophysics Data System (ADS)

Ion-molecule reactions between nucleic acid anions, [M-nH]n, formed via electrospray ionization, and trimethylsilylchloride have been investigated in an ion trap mass spectrometer at a helium bath gas pressure of 1 mtorr. Three types of reactions are observed: (i) SN2(Si) when n > 1 ; (ii) adduct formation when n = 1 ; and (iii) addition followed by elimination of HCl when n = 1 and where an acidic phosphate proton is present (e.g., 5'-pdA). The kinetics of these reactions have been studied for various anions derived from the following deoxyadenosine species: 5'-pdA; 5'-pppdA, 5'-d(AA)-3'; 5'-d(AAA)-3' and 5'-d(AAAA)-3'. The following reactivity order is observed: [M-2H]2- of 5'-pppdA > [M-2H]2- of 5'-d(AAA)-3' > [M-3H]3- of 5'-d(AAAA)-3' > [M-3H + TMS]2- of 5'-d(AAAA)-3' > [M-2H]2- of 5'-d(AAAA)-3' > [M-H]- of 5'-pdA >> [M-H]- of 5'-d(AA)-3' > [M-H]- of 5'-d(AAA)-3'. In addition, the collision-induced dissociation reactions of the products of these reactions have been studied. Decomposition reactions are consistent with trimethylsilyl attachment on the phosphodiester linkage(s) in oligonucleotides and on the phosphate moieties of 5'-pdA and 5'-pppdA. Comparison of data acquired for modified and unmodified oligonucleotide anions of the same charge state reveal that TMS modification can significantly alter the favored dissociation channels, giving rise to sequence information. The results suggest that gas phase TMS derivatization of oligonucleotide anions, combined with tandem mass spectrometry, can provide sequence information complementary to that derived from unmodified anions.

O'Hair, Richard A. J.; McLuckey, Scott A.

1997-03-01

271

Artificial Specific Binders Directly Recovered from Chemically Modified Nucleic Acid Libraries  

PubMed Central

Specific binders comprised of nucleic acids, that is, RNA/DNA aptamers, are attractive functional biopolymers owing to their potential broad application in medicine, food hygiene, environmental analysis, and biological research. Despite the large number of reports on selection of natural DNA/RNA aptamers, there are not many examples of direct screening of chemically modified nucleic acid aptamers. This is because of (i) the inferior efficiency and accuracy of polymerase reactions involving transcription/reverse-transcription of modified nucleotides compared with those of natural nucleotides, (ii) technical difficulties and additional time and effort required when using modified nucleic acid libraries, and (iii) ambiguous efficacies of chemical modifications in binding properties until recently; in contrast, the effects of chemical modifications on biostability are well studied using various nucleotide analogs. Although reports on the direct screening of a modified nucleic acid library remain in the minority, chemical modifications would be essential when further functional expansion of nucleic acid aptamers, in particular for medical and biological uses, is considered. This paper focuses on enzymatic production of chemically modified nucleic acids and their application to random screenings. In addition, recent advances and possible future research are also described. PMID:23094139

Kasahara, Yuuya; Kuwahara, Masayasu

2012-01-01

272

Stability of free and mineral-protected nucleic acids: Implications for the RNA world  

NASA Astrophysics Data System (ADS)

Using molecular dynamics simulations we study the structural stability of three different nucleic acids intercalated within a magnesium aluminium layered double hydroxide (LDH) mineral, at varying degrees of hydration, and free in aqueous solution. The nucleotides investigated are ribose nucleic acid (RNA), deoxyribose nucleic acid (DNA) and peptide nucleic acid (PNA), all in duplex form. Our simulations show that DNA has enhanced Watson-Crick hydrogen-bonding when intercalated within the LDH clay interlayers, compared with intercalated RNA and PNA, whilst the reverse trend is found for the nucleic acids in bulk water. The tendency for LDH to alter the stability of the three nucleic acids persists for higher temperature and pressure conditions. The uncharged protein backbone of PNA is found to have a detrimental effect on the overall stability of the duplex, as it experiences a greatly reduced electrostatic interaction with the charged LDH sheets compared to RNA and DNA. Assuming an RNA world, in which RNA preceded the DNA/protein world, at some point in time DNA must have taken over the role as the information storage molecule from RNA. These results suggest that a mineral based origin of life may have favoured DNA as the information-storage biomolecule over potentially competing RNA and PNA, providing a route to modern biology from the RNA world.

Swadling, Jacob B.; Coveney, Peter V.; Christopher Greenwell, H.

2012-04-01

273

Oxidative Stress and Nucleic Acid Oxidation in Patients with Chronic Kidney Disease  

PubMed Central

Patients with chronic kidney disease (CKD) have high cardiovascular mortality and morbidity and a high risk for developing malignancy. Excessive oxidative stress is thought to play a major role in elevating these risks by increasing oxidative nucleic acid damage. Oxidative stress results from an imbalance between reactive oxygen/nitrogen species (RONS) production and antioxidant defense mechanisms and can cause vascular and tissue injuries as well as nucleic acid damage in CKD patients. The increased production of RONS, impaired nonenzymatic or enzymatic antioxidant defense mechanisms, and other risk factors including gene polymorphisms, uremic toxins (indoxyl sulfate), deficiency of arylesterase/paraoxonase, hyperhomocysteinemia, dialysis-associated membrane bioincompatibility, and endotoxin in patients with CKD can inhibit normal cell function by damaging cell lipids, arachidonic acid derivatives, carbohydrates, proteins, amino acids, and nucleic acids. Several clinical biomarkers and techniques have been used to detect the antioxidant status and oxidative stress/oxidative nucleic acid damage associated with long-term complications such as inflammation, atherosclerosis, amyloidosis, and malignancy in CKD patients. Antioxidant therapies have been studied to reduce the oxidative stress and nucleic acid oxidation in patients with CKD, including alpha-tocopherol, N-acetylcysteine, ascorbic acid, glutathione, folic acid, bardoxolone methyl, angiotensin-converting enzyme inhibitor, and providing better dialysis strategies. This paper provides an overview of radical production, antioxidant defence, pathogenesis and biomarkers of oxidative stress in patients with CKD, and possible antioxidant therapies. PMID:24058721

Sung, Chih-Chien; Hsu, Yu-Chuan; Lin, Yuh-Feng

2013-01-01

274

Polarizable model potential function for nucleic acid bases.  

PubMed

A polarizable model potential (PMP) function for adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) is developed on the basis of ab initio molecular orbital calculations at the MP2/6-31+G* level. The PMP function consists of Coulomb, van der Waals, and polarization terms. The permanent atomic charges of the Coulomb term are determined by using electrostatic potential (ESP) optimization. The multicenter polarizabilities of the polarization term are determined by using polarized one-electron potential (POP) optimization in which the electron density changes induced by a test charge are target. Isotropic and anisotropic polarizabilities are adopted as the multicenter polarizabilities. In the PMP calculations using the optimized parameters, the interaction energies of Watson-Crick type A-T and C-G base pairs were -15.6 and -29.4 kcal/mol, respectively. The interaction energy of Hoogsteen type A-T base pair was -17.8 kcal/mol. These results reproduce well the quantum chemistry calculations at the MP2/6-311++G(3df,2pd) level within the differences of 0.6 kcal/mol. The stacking energies of A-T and C-G were -9.7 and -10.9 kcal/mol. These reproduce well the calculation results at the MP2/6-311++G (2d,2p) level within the differences of 1.3 kcal/mol. The potential energy surfaces of the system in which a sodium ion or a chloride ion is adjacent to the nucleic acid base are calculated. The interaction energies of the PMP function reproduced well the calculation results at the MP2/6-31+G* or MP2/6-311++G(2d,2p) level. The reason why the PMP function reproduces well the high-level quantum mechanical interaction energies is addressed from the viewpoint of each energy terms. PMID:17342710

Nakagawa, Setsuko

2007-07-15

275

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

PubMed

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

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

2014-11-25

276

Variables influencing extraction of nucleic acids from microbial plankton (viruses, bacteria, and protists) collected on nanoporous aluminum oxide filters.  

PubMed

Anodic aluminum oxide (AAO) filters have high porosity and can be manufactured with a pore size that is small enough to quantitatively capture viruses. These properties make the filters potentially useful for harvesting total microbial communities from water samples for molecular analyses, but their performance for nucleic acid extraction has not been systematically or quantitatively evaluated. In this study, we characterized the flux of water through commercially produced nanoporous (0.02 ?m) AAO filters (Anotop; Whatman) and used isolates (a virus, a bacterium, and a protist) and natural seawater samples to test variables that we expected would influence the efficiency with which nucleic acids are recovered from the filters. Extraction chemistry had a significant effect on DNA yield, and back flushing the filters during extraction was found to improve yields of high-molecular-weight DNA. Using the back-flush protocol, the mass of DNA recovered from microorganisms collected on AAO filters was ? 100% of that extracted from pellets of cells and viruses and 94% ± 9% of that obtained by direct extraction of a liquid bacterial culture. The latter is a minimum estimate of the relative recovery of microbial DNA, since liquid cultures include dissolved nucleic acids that are retained inefficiently by the filter. In conclusion, we demonstrate that nucleic acids can be extracted from microorganisms on AAO filters with an efficiency similar to that achievable by direct extraction of microbes in suspension or in pellets. These filters are therefore a convenient means by which to harvest total microbial communities from multiple aqueous samples in parallel for subsequent molecular analyses. PMID:24747903

Mueller, Jaclyn A; Culley, Alexander I; Steward, Grieg F

2014-07-01

277

Surface-Mediated Nucleic Acid Delivery by Lipoplexes Prepared in Microwell Arrays  

PubMed Central

Many delivery methods have been developed to improve the therapeutic efficacy and facilitate the clinical translation of nucleic acid-based therapeutics. A facile surface-mediated nucleic acid delivery by lipoplexes is prepared in a microwell array, which combines the advantages of lipoplexes as an efficient carrier system, surface-mediated delivery, and the control of surface topography. Uniform disc-like lipoplexes containing nucleic acids are formed in the microwell array with a diameter of ~ 818 nm and thickness of ~ 195 nm. The microwell array-mediated delivery of lipoplexes containing FAM-oligodeoxynucleotides is ~ 18.6 and ~ 10.6 times more efficient than the conventional transfection method in an adherent cell line (A549 non-small cell lung cancer cells) and a suspension cell line (KG-1a acute myelogenous leukemia cells), respectively. MicroRNA-29b is then used as a model nucleic acid to investigate the therapeutic efficacy of lipoplexes delivered by the microwell array. Compared to conventional transfection methods, the effective therapeutic dosage of microRNA-29b is reduced from the microgram level to the nanogram level by lipoplexes prepared in the microwell array. The microwell array is also a very flexible platform. Both nucleic acid therapeutics and imaging reagents are incorporated in lipoplexes and successfully delivered to A549 cells, demonstrating its potential applications in theranostic medicine. PMID:23471869

Wu, Yun; Terp, Megan Cavanaugh; Kwak, Kwang Joo; Gallego-Perez, Daniel; Nana-Sinkam, Serge P.; Lee, L. James

2014-01-01

278

Adsorption of Nucleic Acid Components on Rutile (TiO2) Surfaces  

NASA Astrophysics Data System (ADS)

Nucleic acids, the storage molecules of genetic information, are composed of repeating polymers of ribonucleotides (in RNA) or deoxyribonucleotides (in DNA), which are themselves composed of a phosphate moiety, a sugar moiety, and a nitrogenous base. The interactions between these components and mineral surfaces are important because there is a tremendous flux of nucleic acids in the environment due to cell death and horizontal gene transfer. The adsorption of mono-, oligo-, and polynucleotides and their components on mineral surfaces may have been important for the origin of life. We have studied here interactions of nucleic acid components with rutile (TiO2), a mineral common in many terrestrial crustal rocks. Our results suggest roles for several nucleic acid functional groups (including sugar hydroxyl groups, the phosphate group, and extracyclic functional groups on the bases) in binding, in agreement with results obtained from studies of other minerals. In contrast with recent studies of nucleotide adsorption on ZnO, aluminum oxides, and hematite, our results suggest a different preferred orientation for the monomers on rutile surfaces. The conformations of the molecules bound to rutile surfaces appear to favor specific interactions, which in turn may allow identification of the most favorable mineral surfaces for nucleic acid adsorption.

Cleaves, H. James; Jonsson, Caroline M.; Jonsson, Christopher L.; Sverjensky, Dimitri A.; Hazen, Robert M.

2010-04-01

279

Surface-mediated nucleic acid delivery by lipoplexes prepared in microwell arrays.  

PubMed

Many delivery methods have been developed to improve the therapeutic efficacy and facilitate the clinical translation of nucleic acid-based therapeutics. A facile surface-mediated nucleic acid delivery by lipoplexes is prepared in a microwell array, which combines the advantages of lipoplexes as an efficient carrier system, surface-mediated delivery, and the control of surface topography. Uniform disc-like lipoplexes containing nucleic acids are formed in the microwell array with a diameter of ?818 nm and thickness of ?195 nm. The microwell array-mediated delivery of lipoplexes containing FAM-oligodeoxynucleotides is ?18.6 and ?10.6 times more efficient than the conventional transfection method in an adherent cell line (A549 non-small cell lung cancer cells) and a suspension cell line (KG-1a acute myelogenous leukemia cells), respectively. MicroRNA-29b is then used as a model nucleic acid to investigate the therapeutic efficacy of lipoplexes delivered by the microwell array. Compared to conventional transfection methods, the effective therapeutic dosage of microRNA-29b is reduced from the microgram level to the nanogram level by lipoplexes prepared in the microwell array. The microwell array is also a very flexible platform. Both nucleic acid therapeutics and imaging reagents are incorporated in lipoplexes and successfully delivered to A549 cells, demonstrating its potential applications in theranostic medicine. PMID:23471869

Wu, Yun; Terp, Megan Cavanaugh; Kwak, Kwang Joo; Gallego-Perez, Daniel; Nana-Sinkam, Serge P; Lee, L James

2013-07-01

280

Endosomal TLR signaling is required for anti-nucleic acid and rheumatoid factor autoantibodies in lupus  

PubMed Central

Using the Unc93b1 3d mutation that selectively abolishes nucleic acid-binding Toll-like receptor (TLR) (TLR3, -7, -9) signaling, we show these endosomal TLRs are required for optimal production of IgG autoAbs, IgM rheumatoid factor, and other clinical parameters of disease in 2 lupus strains, B6-Faslpr and BXSB. Strikingly, treatment with lipid A, an autoAb-inducing TLR4 agonist, could not overcome this requirement. The 3d mutation slightly reduced complete Freund's adjuvant (CFA)-mediated antigen presentation, but did not affect T-independent type 1 or alum-mediated T-dependent humoral responses or TLR-independent IFN production induced by cytoplasmic nucleic acids. These findings suggest that nucleic acid-sensing TLRs might act as an Achilles' heel in susceptible individuals by providing a critical pathway by which relative tolerance for nucleic acid-containing antigens is breached and systemic autoimmunity ensues. Importantly, this helps provide an explanation for the high frequency of anti-nucleic acid Abs in lupus-like systemic autoimmunity. PMID:19574451

Kono, Dwight H.; Haraldsson, M. Katarina; Lawson, Brian R.; Pollard, K. Michael; Koh, Yi Ting; Du, Xin; Arnold, Carrie N.; Baccala, Roberto; Silverman, Gregg J.; Beutler, Bruce A.; Theofilopoulos, Argyrios N.

2009-01-01

281

Influence of Plasma Processing on Recovery and Analysis of Circulating Nucleic Acids  

PubMed Central

Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp® DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (? = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA. PMID:24205045

Zahra, Nathalie; Primrose, Lindsay; Elshaw, Shona R.; Pringle, J. Howard; Blighe, Kevin; Marchese, Stephanie D.; Hills, Allison; Woodley, Laura; Stebbing, Justin; Coombes, R. Charles; Shaw, Jacqueline A.

2013-01-01

282

Relaxation-Optimized NMR Spectroscopy of Methylene Groups in Proteins and Nucleic Acids  

E-print Network

can be incorporated in many of today's most common 2D and 3D NMR experiments. As an example, we showRelaxation-Optimized NMR Spectroscopy of Methylene Groups in Proteins and Nucleic Acids Emeric acids is of the methylene type. Their detailed study, however, in terms of structure and dynamics by NMR

Clore, G. Marius

283

Supplementary Information An In Vitro Translation, Selection, and Amplification System for Peptide Nucleic Acids  

E-print Network

% trifluoroacetic acid in water for 10 min at room temperature; (iv) reverse-phase HPLC purification of the MMT for Peptide Nucleic Acids Yevgeny Brudno, Michael E. Birnbaum, Ralph E. Kleiner, David R. Liu* Department experiments in main text Figures 2 and 3 and Supplementary Figure 1 were synthesized with a 5'-MMT-amino- d

Liu, David R.

284

Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif.  

PubMed Central

We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis. Images PMID:2885805

Grange, T; de Sa, C M; Oddos, J; Pictet, R

1987-01-01

285

Advances in polymeric and inorganic vectors for nonviral nucleic acid delivery  

PubMed Central

Nonviral systems for nucleic acid delivery offer a host of potential advantages compared with viruses, including reduced toxicity and immunogenicity, increased ease of production and less stringent vector size limitations, but remain far less efficient than their viral counterparts. In this article we review recent advances in the delivery of nucleic acids using polymeric and inorganic vectors. We discuss the wide range of materials being designed and evaluated for these purposes while considering the physical requirements and barriers to entry that these agents face and reviewing recent novel approaches towards improving delivery with respect to each of these barriers. Furthermore, we provide a brief overview of past and ongoing nonviral gene therapy clinical trials. We conclude with a discussion of multifunctional nucleic acid carriers and future directions. PMID:22826857

Sunshine, Joel C; Bishop, Corey J; Green, Jordan J

2014-01-01

286

Beyond DNA origami: A look on the bright future of nucleic acid nanotechnology  

PubMed Central

Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA ”staples”. This revolutionary approach has led to the creation of a multitude of 2D and 3D scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy. PMID:22131292

Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J.

2012-01-01

287

Effect of Diet on Amino and Nucleic Acids of Rumen Bacteria and Protozoa1  

Microsoft Academic Search

Amino acid composition and nucleic acid content of pure cultures of rumen bacteria (17 species) were analyzed. Amino acid composition between gram- positive and -negative organisms was not different. The total nitrogen content of gram-negative bacteria (10.8%) was signif- icantly higher than gram-positive or- ganisms (9.9%). Deoxyribonucleic acid- nitrogen:total nitrogen (rag\\/g) differed between gram-positive (8.8) and gram- negative (18.9) bacteria,

M. J. Arambel; E. E. Bartley; G. S. Dufva; T. G. Nagaraja; A. D. Dayton

1982-01-01

288

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

PubMed

In this article, we describe a disposable nucleic acid biosensor (DNAB) for low-cost and sensitive detection of nucleic acid samples in 15 min. Combining the unique optical properties of gold nanoparticles (Au-NP) and the high efficiency of chromatographic separation, sandwich-type DNA hybridization reactions were realized on the lateral flow strips, which avoid multiple incubation, separation, and washing steps in the conventional nucleic acid biosensors. The captured Au-NP probes on the test zone and control zone of the biosensor produced the characteristic red bands, enabling visual detection of nucleic acid samples without instrumentation. The quantitative detection was performed by reading the intensities of the produced red bands with a portable strip reader. The parameters (e.g., the concentration of reporter probe, the size of Au-NP, the amount of Au-NP-DNA probe, lateral flow membranes, and the concentration of running buffer) that govern the sensitivity and reproducibility of the sensor were optimized. The response of the optimized device is highly linear over the range of 1-100 nM target DNA, and the limit of detection is estimated to be 0.5 nM in association with a 15 min assay time. The sensitivity of the biosensor was further enhanced by using horseradish peroxidase (HRP)-Au-NP dual labels which ensure a quite low detection limit of 50 pM. The DNAB has been applied for the detection of human genomic DNA directly with a detection limit of 2.5 microg/mL (1.25 fM) by adopting well-designed DNA probes. The new nucleic acid biosensor thus provides a rapid, sensitive, low cost, and quantitative tool for the detection of nucleic acid samples. It shows great promise for in-field and point-of-care diagnosis of genetic diseases and detection of infectious agents or warning against biowarfare agents. PMID:19159221

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

2009-02-15

289

Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time  

PubMed Central

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 simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings. PMID:21152399

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

2010-01-01

290

Assessment of methods for covalent binding of nucleic acids to magnetic beads, Dynabeads, and the characteristics of the bound nucleic acids in hybridization reactions.  

PubMed Central

Dynabeads are magnetic monosized beads with high stability, high uniformity, unique paramagnetic properties, low particle-particle interaction, and high dispersibility. Different reactive groups; hydroxyl, carboxyl and amino groups can be attached to the surface. Several methods for covalent attachment of DNA or oligonucleotides to the beads were investigated. Best coupling yields were obtained by carbodiimide-mediated end-attachment of 5'-phosphate and 5'-NH2 modified nucleic acids to respectively amino and carboxyl beads. The carboxyl beads showed a low degree of non-specific binding, while a better yield of end-attached nucleic acids was obtained using the amino beads. The DNA-beads worked efficiently in hybridization experiments, and the kinetics of hybridization approach those of solution hybridization. PMID:3205723

Lund, V; Schmid, R; Rickwood, D; Hornes, E

1988-01-01

291

Identification of Dekkera bruxellensis (Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes  

PubMed Central

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity. PMID:11157265

Stender, Henrik; Kurtzman, Cletus; Hyldig-Nielsen, Jens J.; S?rensen, Ditte; Broomer, Adam; Oliveira, Kenneth; Perry-O'Keefe, Heather; Sage, Andrew; Young, Barbara; Coull, James

2001-01-01

292

Highly selective and sensitive nucleic acid detection based on polysaccharide-functionalized silver nanoparticles.  

PubMed

Polysaccharide-functionalized silver nanoparticles (Oc-AgNPs) with a mean diameter of 15 nm were utilized as a novel and effective fluorescence-sensing platform for nucleic acid detection. Tests on the oligonucleotide sequences associated with the human immunodeficiency virus as a model system showed that the Oc-AgNPs effectively absorbed and quenched dye-labeled single-stranded DNA through strong hydrogen bonding interactions and slight electrostatic attractive interactions. The proposed system efficiently differentiated between complementary and mismatched nucleic acid sequences with high selectivity and good reproducibility at room temperature. PMID:24995414

Yan, Jing-Kun; Ma, Hai-Le; Cai, Pan-Fu; Wu, Jian-Yong

2015-01-01

293

Counter-current chromatographic separation of nucleic acid constituents with an extremely hydrophilic solvent system  

PubMed Central

Nucleic acid constituents such as nucleobases, nucleosides and nucleotides were separated by counter-current chromatography using a type J coil planet centrifuge. The separation was performed with an extremely hydrophilic solvent system composed of 1-propanol/800 mM potassium phosphate buffer (pH 7.4) (1 : 1) by eluting the lower aqueous phase at a flow-rate of 0.5 ml/min. Eight kinds of nucleic acid constituents, including UMP, AMP, deoxyAMP, uridine, urasile, 2’ deoxy uridine, adenosine and adenine were well resolved within 170 min. PMID:20362294

Shibusawa, Yoichi; Shoji, Atsushi; Suzuka, Chihiro; Yanagida, Akio; Ito, Yoichiro

2010-01-01

294

In-situ detection of viral nucleic acids using fluorescent probes  

NASA Astrophysics Data System (ADS)

The objective of this work was to develop and improve technologies in cytometry and molecular biology for the specific in situ detection of viral nucleic acids. The major application for this system was the detection and measurement of individual cells stained specifically for the Human Immunodeficiency Virus (HIV) in patients with Acquired Immune Deficiency Syndrome (AIDS). Staining procedures used nucleic acid either directly or indirect labeled with enzymes or fluorescent probes. A cytometry system was used to acquire digitized images of labeled cells and determine their individual staining density or intensity. Efforts are underway to improve the sensitivity of these assays using time-resolved methods.

Donovan, Richard M.

1990-07-01

295

Ordered Self-Assembled Monolayers of Peptide Nucleic Acids with DNA Recognition Capability  

NASA Astrophysics Data System (ADS)

We report on the formation of ordered self-assembled monolayers (SAMs) of single-stranded peptide nucleic acids (ssPNA). In spite of their remarkable length (7nm) thiolated PNAs assemble standing up on gold surfaces similarly to the SAMs of short alkanethiols. SAMs of ssPNA recognize complementary nucleic acids, acting as specific biosensors that discriminate even a point mutation in target ssDNA. These results are obtained by surface characterization techniques that avoid labeling of the target molecule: x-ray photoemission, x-ray absorption and atomic force microscopy.

Briones, C.; Mateo-Marti, E.; Gómez-Navarro, C.; Parro, V.; Román, E.; Martín-Gago, J. A.

2004-11-01

296

Alternative nucleic acid analogues for programmable assembly: hybridization of LNA to PNA.  

PubMed

Complementary locked nucleic acid (LNA) and peptide nucleic acid (PNA) hexamers bind to each other with significantly higher affinity than each binds to DNA, and with far greater affinity than DNA binds to complementary DNA. The hybridization is highly specific with a single mismatch causing decreases in T(m) values ranging from 12 (G/T) to 30 degrees C (A/A). Importantly, the hybridization of an LNA oligomer to a PNA oligomer is unaffected by the ionic strength of the buffer. These properties make the LNA/PNA pair an attractive candidate as a replacement for DNA in programmable assembly. PMID:15792422

Ng, Pei-Sze; Bergstrom, Donald E

2005-01-01

297

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

PubMed Central

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. PMID:22412335

Lui, Clarissa; Cady, Nathaniel C.; Batt, Carl A.

2009-01-01

298

Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer  

DOEpatents

A method for detecting the presence of a target nucleotide or sequence of nucleotides in a nucleic acid is disclosed. The method is comprised of forming an oligonucleotide labeled with two fluorophores on the nucleic acid target site. The doubly labeled oligonucleotide is formed by addition of a singly labeled dideoxynucleoside triphosphate to a singly labeled polynucleotide or by ligation of two singly labeled polynucleotides. Detection of fluorescence resonance energy transfer upon denaturation indicates the presence of the target. Kits are also provided. The method is particularly applicable to genotyping.

Kwok, Pui-Yan (Clayton, MO); Chen, Xiangning (St. Louis, MO)

1999-01-01

299

Depletion of Unwanted Nucleic Acid Templates by Selective Cleavage: LNAzymes, Catalytically Active Oligonucleotides Containing Locked Nucleic Acids, Open a New Window for Detecting Rare Microbial Community Members  

PubMed Central

Many studies of molecular microbial ecology rely on the characterization of microbial communities by PCR amplification, cloning, sequencing, and phylogenetic analysis of genes encoding rRNAs or functional marker enzymes. However, if the established clone libraries are dominated by one or a few sequence types, the cloned diversity is difficult to analyze by random clone sequencing. Here we present a novel approach to deplete unwanted sequence types from complex nucleic acid mixtures prior to cloning and downstream analyses. It employs catalytically active oligonucleotides containing locked nucleic acids (LNAzymes) for the specific cleavage of selected RNA targets. When combined with in vitro transcription and reverse transcriptase PCR, this LNAzyme-based technique can be used with DNA or RNA extracts from microbial communities. The simultaneous application of more than one specific LNAzyme allows the concurrent depletion of different sequence types from the same nucleic acid preparation. This new method was evaluated with defined mixtures of cloned 16S rRNA genes and then used to identify accompanying bacteria in an enrichment culture dominated by the nitrite oxidizer “Candidatus Nitrospira defluvii.” In silico analysis revealed that the majority of publicly deposited rRNA-targeted oligonucleotide probes may be used as specific LNAzymes with no or only minor sequence modifications. This efficient and cost-effective approach will greatly facilitate tasks such as the identification of microbial symbionts in nucleic acid preparations dominated by plastid or mitochondrial rRNA genes from eukaryotic hosts, the detection of contaminants in microbial cultures, and the analysis of rare organisms in microbial communities of highly uneven composition. PMID:23263968

Dolinsek, Jan; Dorninger, Christiane; Lagkouvardos, Ilias; Wagner, Michael

2013-01-01

300

Depletion of unwanted nucleic acid templates by selective cleavage: LNAzymes, catalytically active oligonucleotides containing locked nucleic acids, open a new window for detecting rare microbial community members.  

PubMed

Many studies of molecular microbial ecology rely on the characterization of microbial communities by PCR amplification, cloning, sequencing, and phylogenetic analysis of genes encoding rRNAs or functional marker enzymes. However, if the established clone libraries are dominated by one or a few sequence types, the cloned diversity is difficult to analyze by random clone sequencing. Here we present a novel approach to deplete unwanted sequence types from complex nucleic acid mixtures prior to cloning and downstream analyses. It employs catalytically active oligonucleotides containing locked nucleic acids (LNAzymes) for the specific cleavage of selected RNA targets. When combined with in vitro transcription and reverse transcriptase PCR, this LNAzyme-based technique can be used with DNA or RNA extracts from microbial communities. The simultaneous application of more than one specific LNAzyme allows the concurrent depletion of different sequence types from the same nucleic acid preparation. This new method was evaluated with defined mixtures of cloned 16S rRNA genes and then used to identify accompanying bacteria in an enrichment culture dominated by the nitrite oxidizer "Candidatus Nitrospira defluvii." In silico analysis revealed that the majority of publicly deposited rRNA-targeted oligonucleotide probes may be used as specific LNAzymes with no or only minor sequence modifications. This efficient and cost-effective approach will greatly facilitate tasks such as the identification of microbial symbionts in nucleic acid preparations dominated by plastid or mitochondrial rRNA genes from eukaryotic hosts, the detection of contaminants in microbial cultures, and the analysis of rare organisms in microbial communities of highly uneven composition. PMID:23263968

Dolinsek, Jan; Dorninger, Christiane; Lagkouvardos, Ilias; Wagner, Michael; Daims, Holger

2013-03-01

301

Nucleic acid hybridization for detection of cell culture-amplified adenovirus.  

PubMed Central

A number of recombinant plasmids containing genomic segments of adenovirus were constructed. Seven cloned probes, as well as total adenovirus type 2 (Ad2) and Ad16 genomic DNA, were tested by a nucleic acid hybridization technique for sensitivity and specificity in detecting adenoviruses in infected cells. Adenovirus DNA was spotted onto a nitrocellulose filter and hybridized with 32P-labeled DNA probes. The probes, total Ad2 genomic DNA, and plasmid pAd2-H (containing the hexon gene from Ad2 DNA) all detected 10 reference serotypes of five genomic subgroups (A through E) with similar sensitivities. However, plasmid pAd2-H required less preparation time than did total Ad2 DNA. Probes pAd2-F (containing the fiber gene from Ad2) and pAd16-BD (containing the BamHI D fragment from Ad16) hybridized only with reference serotypes from the homologous subgroups (C and B, respectively). Of 101 patient isolates amplified in cells, pAd2-H detected 100% of all isolates from both the homologous and the heterologous subgroups. The detection rates for pAd2-F were 100% (subgroup C) and 3.6% (subgroups A, B, and D), and those for pAd16-BD were 100% (subgroup B) and 9.4% (subgroups A, C, and D). A commercial biotinylated product (Pathogene II) was also included in this study for comparison. Images PMID:3230138

Huang, C; Deibel, R

1988-01-01

302

Ultrafast colorimetric detection of nucleic acids based on the inhibition of the oxidase activity of cerium oxide nanoparticles.  

PubMed

A label-free colorimetric method to detect nucleic acids, which relies on target DNA induced shielding of the oxidase activity of CeO2 NPs, is developed. With this novel strategy, target nucleic acids can be identified within a few minutes and without the need for post-purification of PCR products. PMID:25012452

Kim, Moon Il; Park, Ki Soo; Park, Hyun Gyu

2014-08-28

303

Nucleic acid scavenging polymers inhibit extracellular DNA-mediated innate immune activation without inhibiting anti-viral responses.  

PubMed

Toll-like receptor (TLR) family members, 3, 7 and 9 are key components in initiation and progression of autoimmune disorders such as systemic lupus erythematosus (SLE). These TLRs are often referred to as nucleic acid-sensing TLRs based on their ability to recognize DNAs or RNAs produced by pathogens or damaged cells. During autoimmune disease progression these receptors recognize self nucleic acids as well as self nucleic acid-containing complexes and contribute to inflammatory cytokine production and subsequent enhancement of serum autoantibody levels. We have recently discovered that nucleic-acid scavenging polymers (NASPs) can neutralize the proinflammatory effects of nucleic acids. Here, we begin to explore what effects such NASPs have on normal immune function. We show that such NASPs can inhibit TLR activation without affecting nucleic acid-independent T cell activation. Moreover, we observe that stimulation of immune cells by encapsulated nucleic acids, such as those found in viral particles, is unaffected by NASPs. Thus NASPs only limit the activation of the immune system by accessible extra-cellular nucleic acid and do not engender non-specific immune suppression. These important findings suggest that NASPs represent a new approach toward anti-inflammatory drug development as these agents can potentially be utilized to block overt autoimmune disorders and inflammation while allowing normal immune responses to occur. PMID:23936008

Holl, Eda K; Shumansky, Kara L; Pitoc, George; Ramsburg, Elizabeth; Sullenger, Bruce A

2013-01-01

304

Brnsted Acids The Strongest Isolable Acid**  

E-print Network

Brønsted Acids The Strongest Isolable Acid** Mark Juhasz, Stephan Hoffmann, Evgenii Stoyanov, Kee-Chan Kim, and Christopher A. Reed* Acids based on carborane anions as conjugate bases (Figure 1) are a new class of Brønsted (protic) acids, notable for their "strong yet gentle" qualities.[1] For example

Reed, Christopher A.

305

Theoretical Determination of One-Electron Oxidation Potentials for Nucleic Acid Bases  

E-print Network

Theoretical Determination of One-Electron Oxidation Potentials for Nucleic Acid Bases Brian T University, Detroit, Michigan 48202, United States *S Supporting Information ABSTRACT: The oxidation phase proton affinity/gas phase basicity and an MAE of ca. 0.04 eV for the adiabatic/vertical ionization

Schlegel, H. Bernhard

306

IDT SciTools: a suite for analysis and design of nucleic acid oligomers  

Microsoft Academic Search

DNA and RNA oligomers are used in a myriad of diverse biological and biochemical experiments. These oligonucleotides are designed to have unique biophysical, chemical and hybridization pro- perties. We have created an integrated set of bioinformatics tools that predict the properties of native and chemically modified nucleic acids and assist in their design. Researchers can select PCR primers, probes and

Richard Owczarzy; Andrey V. Tataurov; Yihe Wu; Jeffrey A. Manthey; Kyle A. Mcquisten; Hakeem G. Almabrazi; Kent F. Pedersen; Yuan Lin; Justin Garretson; Neil O. Mcentaggart; Chris A. Sailor; Robert B. Dawson; Andrew S. Peek

2008-01-01

307

NUCPLOT: a program to generate schematic diagrams of protein-nucleic acid interactions  

Microsoft Academic Search

Proteins that bind to DNA are found in all areas of genetic activity within the cell. To help understand how these proteins perform their various functions, it is useful to analyse which residues are involved in binding to the DNA and how they interact with the bases and sugar-phosphate backbone of nucleic acids. Here we describe a program called NUCPLOT

Nicholas M. Luscombe; Roman A. Laskowski; Janet M. Thornton

1997-01-01

308

Thermodynamics of Nucleic Acid "Shape Readout" by an Hongjuan Xi, Erik Davis, Nihar Ranjan, Liang Xue,  

E-print Network

as playing an equally important role in DNA recognition. Competition dialysis, UV, flourescent intercalator conformations. Aminoglycoside antibiotics are well-known chemotherapeu- tic agents that have been in clinical is the site of antibiotic action.8 There are other nucleic acid structural forms such as triplex, quadruplex

Stuart, Steven J.

309

A design for computer nucleic-acid-sequence storage, retrieval, and manipulation  

PubMed Central

We have designed and built a data-base system for the storage of nucleic-acid sequences. The system consists of a data base (“the library”) and software that manages and provides access to that data base (“the Librarian”). PMID:7099972

Schneider, Thomas D.; Stormo, Gary D.; Haemer, Jeffrey S.; Gold, Larry

1982-01-01

310

NUCLEIC ACID SYNTHESIS MEASURM,IEII]S IN SEDIMENT MICROBIAI CO}O{UNITIES  

E-print Network

adenine into RNA ng ATp-l n-'1 fo. sedi-ments" DNA synthesis rates were used to calculate carbon growth rate (p) esti-mated frorn DNA synthesis rates and ATP biomass in. surface sed.iments ranged. fromby NUCLEIC ACID SYNTHESIS MEASURM,IEII]S IN SEDIMENT MICROBIAI CO}O{UNITIES: I

Luther, Douglas S.

311

Streamlined determination of processive run length and mechanochemical coupling of nucleic acid motor activities  

Microsoft Academic Search

Quantitative determination of enzymatic rates, pro- cessivity and mechanochemical coupling is a key aspect in characterizing nucleotide triphosphate (NTP)-driven nucleic acid motor enzymes, for both basic research and technological applications. Here, we present a streamlined analytical method suitable for the determination of all key functional parameters based on measurement of NTP hydroly- sis during interaction of motor enzymes with the

M. Gyimesi; K. Sarlos; I. Derenyi; M. Kovacs

2010-01-01

312

Plant Cell and Viral Helicases: Essential Enzymes for Nucleic Acid Transactions  

Microsoft Academic Search

Helicases are ubiquitous enzymes that catalyze the unwinding of energetically stable duplex DNA (DNA helicases) or duplex RNA secondary structures (RNA helicases) and thus play essential role in all aspects of nucleic acid metabolism. All helicases share the common property of being able to use the energy derived from NTP hydrolysis (usually ATP) to break the hydrogen bonds that hold

Narendra Tuteja

2000-01-01

313

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

NASA Astrophysics Data System (ADS)

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

Hatton, Bryan; Rickard, David

2008-06-01

314

Use of peptide nucleic acids (PNAs) for genotyping by solution and surface methods.  

PubMed

Peptide nucleic acids (PNAs) are synthetic oligonucleotide analogues based on a pseudopeptide backbone that bind complementary DNA or RNA with high affinity and specificity. In this chapter, three PNA-based genotyping assays are described: PCR clamping, fluorescence-based recognition, and microarray platform. The first two methods are performed in solution, while the microarray method uses a solid surface. PMID:24297357

Sforza, Stefano; Tedeschi, Tullia; Bencivenni, Mariangela; Tonelli, Alessandro; Corradini, Roberto; Marchelli, Rosangela

2014-01-01

315

Understanding nonviral nucleic acid delivery with quantum dot-FRET nanosensors  

PubMed Central

Nonviral delivery of nucleic acids is a potentially safe and viable therapeutic modality for inherited and acquired diseases. However, current systems have proven too inefficient for widespread clinical translation. The rational design of improved carriers depends on a quantitative, mechanistic understanding of the rate-limiting barriers to efficient intracellular delivery. Separation of the nucleic acid from the carrier is one of the barriers, which may be analyzed by Förster resonance energy transfer (FRET), a mechanism used to detect interactions between fluorescently labeled molecules. When applied to the molecular components of polymer or lipid-based nanocomplexes, FRET provides information on their complexation status, uptake, release and degradation. Recently, the design of FRET systems incorporating quantum dots as energy donors has led to improved signal stability, allowing prolonged measurements, as well as increased sensitivity, enabling direct detection and the potential for multiplexing. The union of quantum dots and FRET is providing new insights into the mechanisms of nonviral nucleic acid delivery through convergent characterization of delivery barriers, and has the potential to accelerate the design of improved carriers to realize the potential of nucleic acid therapeutics and gene medicine. PMID:22471720

Grigsby, Christopher L; Ho, Yi-Ping; Leong, Kam W

2012-01-01

316

A Standard Reference Frame for the Description of Nucleic Acid Base-pair Geometry  

E-print Network

and Human- Technology and Helen M. Berman and Wilma K. Olson of the Nucleic Acid Database Project (supported (Indian Institute Science, Bangalore), Helen M. Berman (Rutgers University), Stephen K. Burley Westbrook and Helen M. Berman. The optimization of standard base-pair geometry and the calculation

Gerstein, Mark

317

N2P3 phosphoramidate glycerol nucleic acid as a potential alternative genetic Jesse J. Chen  

E-print Network

S1 N2P3 phosphoramidate glycerol nucleic acid as a potential alternative genetic system Jesse J-exchange HPLC analysis of the crude product of npGNA synthesis. Sequence: 3-TTT TTT TTT T-2-T, the underlined

Heller, Eric

318

West Nile virus blood transfusion-related infection despite nucleic acid testing  

Microsoft Academic Search

BACKGROUND: A case of West Nile virus (WNV) encephalitis associated with transfusion of blood that did not react when tested for WNV by minipool (MP) nucleic acid testing (NAT) is described. A Nebraska man developed clinical encephalitis 13 days after surgery and transfusion of 26 blood components. Antibody testing confirmed WNV infection. An investigation was initiated to determine the source

Alexandre Macedo de Oliveira; Brady D. Beecham; Susan P. Montgomery; Robert S. Lanciotti; Jeffrey M. Linnen; Cristina Giachetti; Larry A. Pietrelli; Susan L. Stramer; Thomas J. Safranek

2004-01-01

319

Hydration of Nucleic Acid Bases Studied Using Novel Atom-Atom Potential Functions  

Microsoft Academic Search

New simple atom-atom potential functions for simulating behavior of nucleic acids and their fragments in aqueous solutions are suggested. These functions contain terms which are inversely proportional to the first (electrostatics), sixth (or tenth for the atoms, forming hydrogen bonds) and twelfth (repulsion of all the atoms) powers of interatomic distance. For the refinement of the potential function parameters calculations

V. I. Poltev; T. I. Grokhlina; G. G. Malenkov

1984-01-01

320

Do Electrostatic Interactions Destabilize ProteinNucleic Acid Binding? Sanbo Qin,1  

E-print Network

published online as an accepted preprint. The ``Published Online'' date corresponds to the preprint version January 2007; revised 13 February 2007; accepted 14 February 2007 Published online 26 February 2007 in their conformations and their interactions with proteins. Nucleic acid binding sites on proteins are known

Weston, Ken

321

Molecular recognition of nucleic acids by nucleolipid/dendrimer surface complexes.  

PubMed

We show for the first time that 1,2-dilauroyl-sn-glycero-3-phosphatidyladenosine nucleolipid surface complexes with cationic poly(amidoamine) dendrimers can be used to selectively bind DNA including oligonucleotides. This molecular recognition has high potential for applications involving biomedical and bioanalytic devices as well as drug delivery systems based on nucleic acids. PMID:25246334

Arteta, Marianna Yanez; Berti, Debora; Montis, Costanza; Campbell, Richard A; Clifton, Luke A; Skoda, Maximilian W A; Soltwedel, Olaf; Baglioni, Piero; Nylander, Tommy

2014-10-01

322

Inhibitory properties of nucleic acid-binding ligands on protein synthesis Abba Malinaa  

E-print Network

. By intercalating between base-pairs of DNA, they dis- rupt both the activity of DNA and RNA polymerases, as well initiation. We used this information to identify a novel threading intercalator that inhibits Hepatitis C.V. All rights reserved. Keywords: Protein synthesis inhibition; Nucleic acid intercalator; Translation

Beal, Peter A.

323

Nucleic Acids Research Preprint -1 Proteome Analyst: custom predictions with explanations in a  

E-print Network

Nucleic Acids Research Preprint - 1 Proteome Analyst: custom predictions with explanations in a web-based tool for high-throughput proteome annotations Duane Szafron, Paul Lu , Russell Greiner, David S should be addressed. Tel: 780.492.7760; Fax: 780.492.1071; Email paullu@cs.ualberta.ca ABSTRACT Proteome

Lu, Paul

324

Nucleic Acids Research, 2007, 16 doi:10.1093/nar/gkm949  

E-print Network

Nucleic Acids Research, 2007, 1­6 doi:10.1093/nar/gkm949 TranspoGene and micro TEs located inside protein- coding genes of seven species: human, mouse, chicken, zebrafish, fruit flyGene and micro- TranspoGene databases can be used by researchers interested in the effect of TE insertion

Ast, Gil

325

Inhibition of Translation and Bacterial Growth by Peptide Nucleic Acid Targeted to Ribosomal RNA  

Microsoft Academic Search

Peptide nucleic acid (PNA) is a DNA mimic that has shown considerable promise as a lead compound for developing gene therapeutic drugs. We report that PNAs targeted to functional and accessible sites in ribosomal RNA can inhibit translation in an Escherichia coli cell-free transcription\\/translation system, with 50% reductions caused by nanomolar PNA concentrations. The effect in vitro is quantitatively similar

Liam Good; Peter E. Nielsen

1998-01-01

326

Antiproliferative effect in chronic myeloid leukaemia cells by antisense peptide nucleic acids  

Microsoft Academic Search

Peptide nucleic acid (PNA) is a synthetic DNA analogue that is resistant to nucleases and pro- teases and binds with exceptional affinity to RNA. Because of these properties PNA has the potential to become a powerful therapeutic agent to be used in vivo. Until now, however, the use of PNA in vivo has not been much investigated. Here, we have

Valentina Rapozzi; Brigitte E. A. Burm; Susanna Cogoi; Gijs A. van der Marel; Jacques H. van Boom; Franco Quadrifoglio; Luigi E. Xodo

2002-01-01

327

Targeted Delivery of Plasmid DNA to Myogenic Cells via Transferrin-Conjugated Peptide Nucleic Acid  

Microsoft Academic Search

We describe a novel approach to conjugate a targeting ligand to plasmid DNA without affecting either its supercoiled conformation or its ability to be efficiently transcribed. A 14-mer peptide nucleic acid (PNA) containing lysine and cysteine on each end was designed to target to a unique sequence located at the antibiotic resistance gene of the plasmid. The binding of PNA

Kenneth W. Liang; Eric P. Hoffman; Leaf Huang

2000-01-01

328

Specific gene blockade shows that peptide nucleic acids readily enter neuronal cells in vivo  

Microsoft Academic Search

Peptide nucleic acids (PNAs) are DNA analogs that can hybridize to complementary sequences with high affinity and stability. Here, we report the first evidence of intracellular delivery of PNAs in vivo. Two CNS receptors, an opioid (mu) and a neurotensin (NTR-1), were targeted independently by repeated microinjection of PNAs into the periaqueductal gray. Behavioral responses to neurotensin (antinociception and hypothermia)

Beth M Tyler; Daniel J McCormick; Clark V Hoshall; Christopher L Douglas; Karen Jansen; Benjamin W Lacy; Bernadette Cusack; Elliott Richelson

1998-01-01

329

Detection of point mutation in the p53 gene using a peptide nucleic acid biosensor  

Microsoft Academic Search

A 17-mer peptide nucleic acid (PNA) is used as the recognition layer of an electrochemical biosensor for detecting a specific mutation in the p53 gene. The performance of the PNA-derived biosensor is compared with that of its DNA counterpart. The significantly higher specificity of the PNA probe greatly improves the detection of a single point mutation, found in many types

Joseph Wang; Gustavo Rivas; Xiaohua Cai; Manuel Chicharro; Concepcion Parrado; Narasaiah Dontha; Asher Begleiter; Michael Mowat; Emil Palecek; Peter E. Nielsen

1997-01-01

330

Reduction and oxidation of peptide nucleic acid and DNA at mercury and carbon electrodes  

Microsoft Academic Search

Peptide nucleic acid (PNA) is a DNA mimic that binds strongly and specifically to complementary DNA or RNA oligomers, but in contrast to DNA its backbone does not carry any electric charge. We used voltammetry in cyclic and square-wave modes to study reduction and oxidation signals of single stranded (ss)PNA and DNA decamers and pentadecamers with the same base sequences

Miroslav Tomschik; František Jelen; Lud?k Havran; Libuše Trnková; Peter E Nielsen; Emil Pale?ek

1999-01-01

331

Photochemical Internalization of a Peptide Nucleic Acid Targeting the Catalytic Subunit of Human Telomerase1  

Microsoft Academic Search

Because peptide nucleic acids (PNAs) are poorly taken up by mamma- lian cells, strategies need to be developed for their intracellular delivery. In the present study, we demonstrated the possibility to efficiently release a naked PNA targeting the catalytic component of human telomerase reverse transcriptase (hTERT-PNA) into the cytoplasm of DU145 prostate cancer cells through the photochemical internalization approach. After

Marco Folini; Kristian Berg; Enrico Millo; Raffaella Villa; Lina Prasmickaite; Maria Grazia Daidone; Umberto Benatti; Nadia Zaffaroni

332

Targeting peptide nucleic acid-protein conjugates to structural features within duplex DNA  

Microsoft Academic Search

A convenient small scale synthesis has been developed for obtaining peptide nucleic acid oligomers (PNAs). PNAs have been conjugated to a protein, staphylococcal nuclease, through disulfide exchange between a cysteine at the 3?-(carboxy) end of the PNA and an introduced cysteine on the surface of the nuclease. Site specific DNA cleavage by the attached nuclease has been used to examine

James C. Norton; John H. Waggenspack; Elana Varnum; David R. Corey

1995-01-01

333

Volume 14 Number 22 1986 NucleicAcids Research Multiple sequence alignment by consensus  

E-print Network

, while the two sequenoe case has been adequately solved for many cases, the situation for alignment of whiah we are aware His method 0 IRL Press Limited, Oxford, England. 9095 #12;NucleicAcids Research and Du(1984)(14) oonsider the longest common subsequenoe of a set of strings. authors seem aware

Waterman, Michael S.

334

Clinical applications of aptamers and nucleic acid therapeutics in haematological malignancies.  

PubMed

Haematological malignancies result from a heterogeneous mix of genetic mutations and chromosome aberrations and translocations. Targeted therapies, such as the anti-CD20 antibody rituximab, or the BCR-ABL1 inhibitor imatinib, have proven to be effective treatments in the management of some of these malignancies, though relapsing or refractory disease is still common. Nucleic acid-based therapies have also entered the clinical arena, providing an alternative, complementary approach. The forerunner of these therapies were the antisense oligonucleotides, but their scope has expanded to include short-interfering RNA (siRNA), microRNA, decoy oligonucleotides and aptamers. These can be used either as mono-therapeutics, in conjunction with current chemotherapy regimens, or in combination with each other to improve therapeutic efficacy. Not only can these nucleic acid-based therapies silence target genes, they also have the potential of restoring gene function. While challenges remain in delivering effective doses of nucleic acid in vivo, these are steadily being met, suggesting an optimistic future in the treatment of haematological malignancies. This review summarizes the application of nucleic acid-based therapeutics, particularly aptamers, in the diagnosis and treatment of haematological malignancies. PMID:21810089

Shigdar, Sarah; Ward, Alister C; De, Abhijit; Yang, Chaoyong J; Wei, Mingqian; Duan, Wei

2011-10-01

335

Potential advantages and risks of nucleic acid vaccines for infant immunization  

Microsoft Academic Search

Infant antibody responses are impaired due to defective B cell activation by bacterial polysaccharides, slow B cell responses to protein antigens and reduced T cell helper activities. These features result in a greater susceptibility to severe infections by encapsulated bacteria and the requirement for repeated doses of vaccines during the first years of life. Nucleic acid vaccines could optimize infant

Claire-Anne Siegrist

1997-01-01

336

IN VITRO STUDIES ON LONGWAVE ULTRAVIOLET LIGHT-DEPENDENT REACTIONS OF THE SKIN PHOTOSENSITIZER CHLORPROMAZINE WITH NUCLEIC ACIDS, PURINES AND PYRIMIDINES  

Microsoft Academic Search

Fluorescence spectral shifts were observed when chlorpromazine was irradiated with long-wave ultraviolet light in the presence of nucleic acids. An increase in fluorescence was accompanied by incorporation of radioactivity from 3H-chlorpromazine into the alcohol-insoluble (nucleic acid) fraction of reaction mixtures. Apparent complex formation proceeded at a faster rate with single-stranded nucleic acids than with double-stranded nucleic acids. Spectral shifts also

Guinter Kahn; Barry P. Davis

1970-01-01

337

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

PubMed

Azobenzene has been widely used as a photoregulator due to its reversible photoisomerization, large structural change between E and Z isomers, high photoisomerization yield, and high chemical stability. On the other hand, some azobenzene derivatives can be used as universal quenchers for many fluorophores. Nucleic acid is a good candidate to be modified because it is not only the template of gene expression but also widely used for building well-organized nanostructures and nanodevices. Because the size and polarity distribution of the azobenzene molecule is similar to a nucleobase pair, the introduction of azobenzene into nucleic acids has been shown to be an ingenious molecular design for constructing light-switching biosystems or light-driven nanomachines. Here we review recent advances in azobenzene-modified nucleic acids and their applications for artificial regulation of gene expression and enzymatic reactions, construction of photoresponsive nanostructures and nanodevices, molecular beacons, as well as obtaining structural information using the introduced azobenzene as an internal probe. In particular, nucleic acids bearing multiple azobenzenes can be used as a novel artificial nanomaterial with merits of high sequence specificity, regular duplex structure, and high photoregulation efficiency. The combination of functional groups with biomolecules may further advance the development of chemical biotechnology and biomolecular engineering. PMID:25236334

Li, Jing; Wang, Xingyu; Liang, Xingguo

2014-12-01

338

Sites in nucleic acids reacting with alkylating agents of differing carcinogenicity or mutagenicity  

Microsoft Academic Search

The site of alkylation of a nucleic acid, in vitro or in vivo, is greatly dependent on the type of alkylating agent. Most alkylating agents of low mutagenicity or carcinogenicity (such as dimethylsulfate) react primarily with the ring nitrogens. The carcinogenic N?nitroso compounds have a great affinity for alkylating oxygens and react with all ring oxygens as well as the

B. Singer

1977-01-01

339

ll cells contain lots of big molecules, especially proteins, nucleic acids and  

E-print Network

, the mitochondrion and endoplasmic reticulum -- shows that their diffusion rate is reduced by factors in the range 3A ll cells contain lots of big molecules, especially proteins, nucleic acids and complex sugars effects on cellular processes. The high total concentration of macro- molecules inside cells (up to 400

Weston, Ken

340

Efficient transfer of information from hexitol nucleic acids to RNA during nonenzymatic oligomerization  

NASA Technical Reports Server (NTRS)

Hexitol nucleic acids (HNAs) are DNA analogues that contain the standard nucleoside bases attached to a phosphorylated 1,5-anhydrohexitol backbone. We find that HNAs support efficient information transfer in nonensymatic template-directed reactions. HNA heterosequences appeared to be superior to the corresponding DNA heterosequences in facilitating synthesis of complementary oligonucleotides from nucleoside-5'-phosphoro-2-methyl imidazolides.

Kozlov, I. A.; De Bouvere, B.; Van Aerschot, A.; Herdewijn, P.; Orgel, L. E.

1999-01-01

341

RNA:DNA ratio and other nucleic acid derived indices in marine ecology.  

PubMed

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. PMID:19325815

Chícharo, Maria A; Chícharo, Luis

2008-08-01

342

Helicase proteins DHX29 and RIG-I cosense cytosolic nucleic acids in the human airway system  

PubMed Central

The recognition of cytoplasmic nucleic acid is critical for innate immune responses against microbial infection and is responsible for autoimmunity induced by dead cells. Here, we report the identification of a unique cytosolic nucleic acid cosensor in human airway epithelial cells and fibroblasts: DEAH (Asp-Glu-Ala-His) box polypeptide 29 (DHX29), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family. Knocking down DHX29 by siRNA attenuated the ability of cells to mount type I IFN and IL-6 in response to cytosolic nucleic acids and various viruses by blocking the activation of interferon regulatory factor 3 and NF-?B-p65. The cytosolic nucleic acid sensing by DHX29 in human epithelial cells and fibroblasts is independent of stimulator of interferon genes but is dependent on retinoic acid-inducible gene 1 (RIG-I) and mitochondrial antiviral signaling protein (MAVS). DHX29 binds directly to nucleic acids and interacts with RIG-I and MAVS through its helicase 1 domain, activating the RIG-I–MAVS-dependent cytosolic nucleic acid response. These results suggest that DHX29 is a cytosolic nucleic acid cosensor that triggers RIG-I/MAVS-dependent signaling pathways. This study will have important implications in drug and vaccine design for control of viral infections and viral-induced pathology in the airway. PMID:24821782

Sugimoto, Naoshi; Mitoma, Hiroki; Kim, Taeil; Hanabuchi, Shino; Liu, Yong-Jun

2014-01-01

343

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

NASA Technical Reports Server (NTRS)

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 polymers with related peptide backbones were the dominant information macromolecules.

Nelson, Kevin E.; Miller, Stanley L.

1992-01-01

344

Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using  

DOEpatents

A primer directed DNA amplification method to isolate efficiently chromosome-specific repeated DNA wherein degenerate oligonucleotide primers are used is disclosed. The probes produced are a heterogeneous mixture that can be used with blocking DNA as a chromosome-specific staining reagent, and/or the elements of the mixture can be screened for high specificity, size and/or high degree of repetition among other parameters. The degenerate primers are sets of primers that vary in sequence but are substantially complementary to highly repeated nucleic acid sequences, preferably clustered within the template DNA, for example, pericentromeric alpha satellite repeat sequences. The template DNA is preferably chromosome-specific. Exemplary primers and probes are disclosed. The probes of this invention can be used to determine the number of chromosomes of a specific type in metaphase spreads, in germ line and/or somatic cell interphase nuclei, micronuclei and/or in tissue sections. Also provided is a method to select arbitrarily repeat sequence probes that can be screened for chromosome-specificity. 18 figs.

Weier, H.U.G.; Gray, J.W.

1995-06-27

345

Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using  

DOEpatents

A primer directed DNA amplification method to isolate efficiently chromosome-specific repeated DNA wherein degenerate oligonucleotide primers are used is disclosed. The probes produced are a heterogeneous mixture that can be used with blocking DNA as a chromosome-specific staining reagent, and/or the elements of the mixture can be screened for high specificity, size and/or high degree of repetition among other parameters. The degenerate primers are sets of primers that vary in sequence but are substantially complementary to highly repeated nucleic acid sequences, preferably clustered within the template DNA, for example, pericentromeric alpha satellite repeat sequences. The template DNA is preferably chromosome-specific. Exemplary primers ard probes are disclosed. The probes of this invention can be used to determine the number of chromosomes of a specific type in metaphase spreads, in germ line and/or somatic cell interphase nuclei, micronuclei and/or in tissue sections. Also provided is a method to select arbitrarily repeat sequence probes that can be screened for chromosome-specificity.

Weier, Heinz-Ulrich G. (Tracy, CA); Gray, Joe W. (San Francisco, CA)

1995-01-01

346

A nucleic acid-based test for detection of Fasciola hepatica.  

PubMed

The use of nucleic acid techniques in the diagnosis of parasitic infection has become increasingly widespread. An oligonucleotide probe derived from a rRNA sequence was developed for the detection of Fasciola hepatica in its intermediate snail host Pseudosuccinea columella. Total RNA obtained from whole adult liver flukes was used in a polymerase chain reaction to isolate and amplify a region of approximately 650 base pairs in the small subunit rRNA. This portion of the ribosomal cDNA, which contains highly conserved regions as well as variable regions, was subcloned and sequenced. In comparison to known small subunit rRNA sequences, a sequence unique to F. hepatica was identified and an oligonucleotide probe (CS4) for detection of F. hepatica was developed. A northern blot analysis using CS4 successfully identified small subunit rRNA from F. hepatica. Slot-blot analysis determined that RNA derived from 5 miracidia can be detected with CS4. Moreover, a slot blot utilizing CS4 distinguished RNA derived from snails infected with F. hepatica from RNA of uninfected snails. PMID:1403423

Shubkin, C D; White, M W; Abrahamsen, M S; Rognlie, M C; Knapp, S E

1992-10-01

347

Growth and Synthesis of Nucleic Acid and Protein by Excised Radish Cotyledons 1  

PubMed Central

Nutritional and light requirements for growth and synthesis of RNA, DNA, and protein by cotyledons excised from 5-day-old seedlings of Raphanus sativus L. were investigated, and the course of synthesis was followed through the cell cycle. The minimum requirements for a net increase in nucleic acid and protein were sugar, nitrate, and light. The cotyledons used nitrite at low concentration, but not ammonium ion. Light was required for preliminary steps in synthesis of RNA, DNA, and protein, but the actual polymerization reactions occurred in the dark. The cotyledons contained sufficient endogenous growth factors for about half of the cells to complete 1 cycle on a medium of 1% sucrose, 80 mm KNO3. The increase in DNA was limited to about 50% and was accompanied by a comparable increase in cell number. Fresh weight, RNA, and protein tended to increase in proportion to DNA. Growth of the isolated cotyledons commenced with cell enlargement. RNA began to increase after about 4 hours, DNA after about 12. The major increase in protein also began at about 12 hours. The maximum rate of increase for all 3 occurred between 12 and 16 hours. Cell counts indicated that by 28 hours most of the cells which had replicated DNA had also completed cell division. PMID:16656601

Nieman, R. H.; Poulsen, L. L.

1967-01-01

348

Nucleic Acid Dipstick Test for Molecular Diagnosis of Pandemic H1N1 ?  

PubMed Central

A new nucleic acid amplification-based rapid test for diagnosis of pandemic influenza (H1N1) 2009 virus was developed. The molecular test for pandemic H1N1, SAMBA (simple amplification-based assay), is based on isothermal amplification and visual detection on a dipstick characterized by high sensitivity, high specificity, a short turnaround time, and minimal technical requirements. The amplification step is monitored with an internal control to ensure correct interpretation of test results. The clinical performance of this assay was evaluated using blinded RNA samples extracted from nasal/throat swab specimens from 262 patients exhibiting influenza-like illness. Compared with the United Kingdom National Standard Method, based on quantitative reverse transcription-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the new assay were 95.3% (95% confidence interval, 88.5 to 98.7%), 99.4% (95% confidence interval, 96.9 to 99.9%), 98.8% (95% confidence interval, 93.5 to 99.9%), and 97.8% (95% confidence interval, 94.4 to 99.4%), respectively. The SAMBA for pandemic H1N1 provides a new technology that could potentially facilitate timely diagnosis and management of infected individuals, thereby informing decision making with regard to patient isolation during a pandemic outbreak. PMID:20668123

Wu, Liang-Ta; Curran, Martin D.; Ellis, Joanna S.; Parmar, Surendra; Ritchie, Allyson V.; Sharma, Pia I.; Allain, Jean-Pierre; Jalal, Hamid; Zambon, Maria; Lee, Helen H.

2010-01-01

349

Flow cytometric detection of EBV (EBER snRNA) using peptide nucleic acid probes  

Microsoft Academic Search

The application of peptide nucleic acid (PNA) probes for detection of Epstein-Barr Virus (EBV) snRNA in fixed cells is described. Fluorescein labelled PNA probes were used to detect EBER1 and EBER2 snRNA in Raji, Daudi and HS-Sultan cells. The fixation and permeabilization of cells were optimized. The optimal fixation was found to be 5% acetic acid plus 4% paraformaldehyde in

Tom Just; Heidi Burgwald; Marianne Kjærvig Broe

1998-01-01

350

Detection of human enteric viruses in oysters by in vivo and in vitro amplification of nucleic acids.  

PubMed Central

This study describes the detection of enteroviruses and hepatitis A virus in 31 naturally contaminated oyster specimens by nucleic acid amplification and oligonucleotide probing. Viruses were extracted by adsorption-elution-precipitation from 50-g oyster samples harvested from an area receiving sewage effluent discharge. Ninety percent of each extract was inoculated into primate kidney cell cultures for virus isolation and infectivity assay. Viruses in the remaining 10% of oyster extract that was not inoculated into cell cultures were further purified and concentrated by a procedure involving Freon extraction, polyethylene glycol precipitation, and Pro-Cipitate precipitation. After 3 to 4 weeks of incubation, RNA was extracted from inoculated cultures that were negative for cytopathic effects (CPE). These RNA extracts and the RNA from virions purified and concentrated directly from oyster extracts were subjected to reverse transcriptase PCR (RT-PCR) with primer pairs for human enteroviruses and hepatitis A virus. The resulting amplicons were confirmed by internal oligonucleotide probe hybridization. For the portions of oyster sample extracts inoculated into cell cultures, 12 (39%) were positive for human enteroviruses by CPE and 6 (19%) were positive by RT-PCR and oligoprobing of RNA extracts from CPE-negative cell cultures. For the remaining sample portions tested by direct RT-PCR and oligoprobing after further concentration, five (about 16%) were confirmed to be positive for human enteroviruses. Hepatitis A virus was also detected in RNA extracts of two CPE-positive samples by RT-PCR and oligoprobing. Combining the data from all three methods, enteric viruses were detected in 18 of 31 (58%) samples. Detection by nucleic acid methods increased the number of positive samples by 50% over detection by CPE in cell culture. Hence, nucleic acid amplification methods increase the detection of noncytopathic human enteric viruses in oysters. PMID:8837433

Chung, H; Jaykus, L A; Sobsey, M D

1996-01-01

351

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

NASA Astrophysics Data System (ADS)

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

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

2010-02-01

352

ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids  

PubMed Central

It is informative to detect highly conserved positions in proteins and nucleic acid sequence/structure since they are often indicative of structural and/or functional importance. ConSurf (http://consurf.tau.ac.il) and ConSeq (http://conseq.tau.ac.il) are two well-established web servers for calculating the evolutionary conservation of amino acid positions in proteins using an empirical Bayesian inference, starting from protein structure and sequence, respectively. Here, we present the new version of the ConSurf web server that combines the two independent servers, providing an easier and more intuitive step-by-step interface, while offering the user more flexibility during the process. In addition, the new version of ConSurf calculates the evolutionary rates for nucleic acid sequences. The new version is freely available at: http://consurf.tau.ac.il/. PMID:20478830

Ashkenazy, Haim; Erez, Elana; Martz, Eric; Pupko, Tal; Ben-Tal, Nir

2010-01-01

353

Nucleic Acid-directed Self-assembly of Multifunctional Gold Nanoparticle Imaging Agents1  

PubMed Central

Gold nanoparticles have attracted much interest as a platform for development of multifunctional imaging and therapeutic agents. Multifunctionalized gold nanoparticles are generally constructed by covalent assembly of a gold core with thiolated ligands. In this study, we have assembled multifunctionalized gold nanoparticles in one step by nucleic acid hybridization of ODN (oligodeoxynucleotide)-derivatized gold nanoparticles with a library of pre-functionalized complementary PNAs (peptide nucleic acids). The PNAs were functionalized by conjugation with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) for chelating 64Cu for PET imaging, PEG (polyethylene glycol) for conferring stealth properties, and Cy5 for fluorescent imaging. The resulting nanoparticles showed good stability both in vitro and in vivo showing biodistribution behavior in a mouse that would be expected for a PEGylated gold nanoparticle rather than that for the radiolabelled PNA used in its assembly. PMID:24058728

Zhang, Ziyan; Liu, Yongjian; Jarreau, Chad; Welch, Michael J.; Taylor, John-Stephen A.

2013-01-01

354

Differentiation of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacterial Liquid Cultures by Using Peptide Nucleic Acid-Fluorescence In Situ Hybridization Probes  

Microsoft Academic Search

A blinded comparison of peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) with routine identification methods was performed on 74 consecutively positive mycobacterial liquid cultures. All Mycobac- terium tuberculosis cultures (48 of 48) and 22 of 27 (81.5%) nontuberculous cultures were correctly identified (including one mixed culture). Five isolates yielded no reaction with either probe and were identified as Mycobacterium xenopi,

F. A. DROBNIEWSKI; P. G. MORE; G. S. HARRIS

2000-01-01

355

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

NASA Technical Reports Server (NTRS)

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

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

1999-01-01

356

Physicochemical and biological characterization of targeted, nucleic acid-containing nanoparticles  

PubMed Central

Nucleic acid-based therapeutics have the potential to provide potent and highly specific treatments for a variety of human ailments. However, systemic delivery continues to be a significant hurdle to success. Multifunctional nanoparticles are being investigated as systemic, nonviral delivery systems, and here we describe the physicochemical and biological characterization of cyclodextrin-containing polycations (CDP) and their nanoparticles formed with nucleic acids including plasmid DNA (pDNA) and small interfering RNA (siRNA). These polycation/nucleic acid complexes can be tuned by formulation conditions to yield particles with sizes ranging from 60–150 nm, zeta potentials from 10–30 mV, and molecular weights from ~7×107–1×109 g mol?1 as determined by light scattering techniques. Inclusion complexes formed between adamantane (AD)-containing molecules and the ?-cyclodextrin molecules enable the modular attachment of polyethylene glycol (AD-PEG) conjugates for steric stabilization and targeting ligands (AD-PEG-transferrin) for cell-specific targeting. A 70-nm particle can contain ~10,000 CDP polymer chains, ~2,000 siRNA molecules, ~4,000 AD-PEG5000 molecules, and ~100 AD-PEG5000-Tf molecules; this represents a significant payload of siRNA and a large ratio of siRNA to targeting ligand (20:1). The particles protect the nucleic acid payload from nuclease degradation, do not aggregate at physiological salt concentrations, and cause minimal erythrocyte aggregation and complement fixation at the concentrations typically used for in vivo application. Uptake of the nucleic acid-containing particles by HeLa cells is measured by flow cytometry and visualized by confocal microscopy. Competitive uptake experiments show that the transferrin-targeted particles display enhanced affinity for the transferrin receptor through avidity effects (multi-ligand binding). Functional efficacy of the delivered pDNA and siRNA is demonstrated through luciferase reporter protein expression and knockdown, respectively. The analysis of the CDP delivery vehicle provides insights that can be applied to the design of targeted nucleic acid delivery vehicles in general. PMID:17326672

Bartlett, Derek W.; Davis, Mark E.

2008-01-01

357

Thermal and Electrical Properties of Nucleic Acids and Proteins  

Microsoft Academic Search

IN 1955, Duchesne and Monfils1 discovered absorption lines in the frequency-range of 30 Mc.\\/sec. in the sodium salts of deoxyribonucleic acid and nucleo-protein of yeast and of ribonucleic acid of Escherichia coli, all in the dry state, when these substances were inserted in the condenser of a super-regenerative oscillator.

J. Duchesne; J. Depireux; A. Bertinchamps; N. Cornet; J. M. van der Kaa

1960-01-01

358

Nucleic Acid Sequence Analysis of Basal Core Promoter\\/Precore\\/Core Region of Hepatitis B Virus Isolated from Chronic Carriers of the Virus from Kolkata, Eastern India: Low Frequency of Mutation in the Precore Region  

Microsoft Academic Search

Objective: The aim of the present study was to characterize the predominant hepatitis B virus (HBV) strains and their molecular variants present in the HBV isolates of the different genotypes found among the chronic carriers of the virus in our community. Methods: Precore\\/core and core promoter regions of HBV DNA were amplified by polymerase chain reaction and then subjected to

Arup Banerjee; Soma Banerjee; Abhijit Chowdhury; Amal Santra; Sujit Chowdhury; Susanta Roychowdhury; Chinmoy Kumar Panda; Sujit Kumar Bhattacharya; Runu Chakravarty

2005-01-01

359

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

PubMed Central

The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K+ rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes exhibits several unique structural features. Five consecutive guanine residues are all involved in G-quartet formation and occupy positions in adjacent DNA strands, which are bridged with a no-residue propeller-type loop. A two-residue D-shaped loop facilitates inclusion of an isolated guanine residue into the vacant spot within the G-quartet. The remaining two G-rich tracts of three residues each adopt parallel orientation and are linked with edgewise and propeller loops. Both 5? with 3 nt and 3? with 4 nt overhangs display well-defined conformations, with latter adopting a basket handle topology. Locked residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex. Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general. PMID:23935071

Marusic, Maja; Veedu, Rakesh N.; Wengel, Jesper; Plavec, Janez

2013-01-01

360

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

PubMed

The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K(+) rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes exhibits several unique structural features. Five consecutive guanine residues are all involved in G-quartet formation and occupy positions in adjacent DNA strands, which are bridged with a no-residue propeller-type loop. A two-residue D-shaped loop facilitates inclusion of an isolated guanine residue into the vacant spot within the G-quartet. The remaining two G-rich tracts of three residues each adopt parallel orientation and are linked with edgewise and propeller loops. Both 5' with 3 nt and 3' with 4 nt overhangs display well-defined conformations, with latter adopting a basket handle topology. Locked residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex. Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general. PMID:23935071

Maruši?, Maja; Veedu, Rakesh N; Wengel, Jesper; Plavec, Janez

2013-11-01

361

Study on the Interaction Between Nucleic Acids and Acetamiprid  

Microsoft Academic Search

The interaction between deoxyribonucleic acid (DNA) and acetamiprid was studied. It was found that the fluorescence of acetamiprid could be enhanced in the presence of DNA in sulfuric acid solution. The excitation and emission wavelength of acetamiprid was 291 nm and 587 nm, respectively. Under optimal conditions, the calibration graph is over the range of 0.1–10 µg mL. The calibration limit is 0.06 µg mL (S\\/N = 3).

Nianqin Jie; Shicong Hou; Fengpei Du; Linrui Huang; Guibin Jiang; Suqin Lv

2003-01-01

362

Original article Comparison of nucleic and amino acid sequences and  

E-print Network

Centre-Ville, Montreal, PQ H3C 3P8; 2Animal Health Laboratory, Laboratory Services Division, Univer American and five Canadian isolates) and the Arvac vaccine strain were compared with those of the Bucyrus the Arvac vaccine strain) and the Bucyrus reference strain ranged from 85.6 to 98.8%, and 85.3 to 98

Boyer, Edmond

363

Gefitinib for non-small-cell lung cancer patients with epidermal growth factor receptor gene mutations screened by peptide nucleic acid-locked nucleic acid PCR clamp  

PubMed Central

This study was prospectively designed to evaluate a phase II study of gefitinib for non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations. Clinical samples were tested for EGFR mutations by peptide nucleic acid-locked nucleic acid PCR clamp, and patients having EGFR mutations were given gefitinib 250?mg daily as the second treatment after chemotherapy. Poor PS patients omitted chemotherapy. Of 107 consecutive patients enrolled, samples from 100 patients were informative, and EGFR mutations were observed in 38 patients. Gefitinib was given to 27 patients with EGFR mutations, and the response rate was 78% (one complete response and 20 partial responses; 95% confidence interval: 58–93%). Median time to progression and median survival time (MST) from gefitinib treatment were 9.4 and 15.4 months, respectively. Grade 3 hepatic toxicity and skin toxicity were observed in one patient each. There were significant differences between EGFR mutations and wild-type patients in response rates (78 vs 14%, P=0.0017), and MST (15.4 vs 11.1 months, P=0.0135). A Cox proportional hazards model indicated that negative EGFR mutation was a secondary prognostic factor (hazards ratio: 2.259, P=0.036). This research showed the need for screening for EGFR mutations in NSCLC patients. PMID:17106442

Sutani, A; Nagai, Y; Udagawa, K; Uchida, Y; Koyama, N; Murayama, Y; Tanaka, T; Miyazawa, H; Nagata, M; Kanazawa, M; Hagiwara, K; Kobayashi, K

2006-01-01

364

Functional nucleic acid-based sensors for heavy metal ion assays.  

PubMed

Heavy metal contaminants such as lead ions (Pb(2+)), mercury ions (Hg(2+)) and silver ions (Ag(+)) can cause significant harm to humans and generate enduring bioaccumulation in ecological systems. Even though a variety of methods have been developed for Pb(2+), Hg(2+) and Ag(+) assays, most of them are usually laborious and time-consuming with poor sensitivity. Due to their unique advantages of excellent catalytic properties and high affinity for heavy metal ions, functional nucleic acids such as DNAzymes and aptamers show great promise in the development of novel sensors for heavy metal ion assays. In this review, we summarize the development of functional nucleic acid-based sensors for the detection of Pb(2+), Hg(2+) and Ag(+), and especially focus on two categories including the direct assay and the amplification-based assay. We highlight the emerging trends in the development of sensitive and selective sensors for heavy metal ion assays as well. PMID:25356810

Zhu, Guichi; Zhang, Chun-Yang

2014-11-10

365

Final Report Nucleic Acid System - PCR, Multiplex Assays and Sample Preparation Project  

SciTech Connect

The objective of this project was to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction). This entailed not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This project had two principal deliverables: (1) design, construct, test and deliver a 24 chamber, multiplex capable suitcase sized PCR instrument, and (2) develop and reduce to practice a multiplex assay for the detection of PCR product by flow cytometry. In addition, significant resources were allocated to test and evaluation of the Hand-held Advanced Nucleic Acid Analyzer (HANAA). This project helps provide the signature and intelligence gathering community the ability to perform, on-site or remote, rapid analysis of environmental or like samples for the presence of a suite of biological warfare pathogens.

Koopman, R.P.; Langlois, R.G.; Nasarabadi, S.; Benett, W.J.; Richards, J.B.; Hadley, D.R.; Miles, R.R.; Brown, S.B.; Stratton, P.L.; Milanovich, F.P.

2001-04-20

366

Loading strategies of ring-shaped nucleic acid translocases and helicases.  

PubMed

Ring-shaped nucleic acid translocases and helicases catalyze the directed and processive movement of nucleic acid strands to support essential transactions such as replication, transcription, and chromosome partitioning. Assembled typically as hexamers, ring helicase/translocase systems use coordinated cycles of nucleoside triphosphate (NTP) hydrolysis to translocate extended DNA or RNA substrates through a central pore. Ring formation presents a topological challenge to the engagement of substrate oligonucleotides, and is frequently overcome by distinct loading strategies for shepherding specific motors onto their respective substrates. Recent structural studies that capture different loading intermediates have begun to reveal how different helicase/translocase rings either assemble around substrates or crack open to allow DNA or RNA strand entry, and how dedicated chaperones facilitate these events in some instances. Both prevailing mechanistic models and remaining knowledge gaps are discussed. PMID:24878340

O'Shea, Valerie L; Berger, James M

2014-04-01

367

Layer-by-layer assembled gold nanoparticles for the delivery of nucleic acids.  

PubMed

The delivery of nucleic acids to mammalian cells requires a potent particulate carrier system. The physicochemical properties of the used particles, such as size and surface charge, strongly influence the cellular uptake and thereby the extent of the subsequent biological effect. However the knowledge of this process is still fragmentary because heterogeneous particle collectives are applied. Therefore we present a strategy to synthesize carriers with a highly specific appearance on the basis of gold nanoparticles (AuNPs) and the Layer-by-Layer (LbL) technique. The LbL method is based on the alternate deposition of oppositely charged (bio-)polymers, in our case poly(ethylenimine) and nucleic acids. The size and surface charge of those particles can be easily modified and accordingly systematic studies on cellular uptake are accessible. PMID:23070770

Wurster, Eva-Christina; Elbakry, Asmaa; Göpferich, Achim; Breunig, Miriam

2013-01-01

368

Integrated Microcapillary for Sample-to-Answer Nucleic Acid Pretreatment, Amplification, and Detection.  

PubMed

This work develops an integrated microcapillary-based loop-mediated isothermal amplification (icLAMP) containing preloaded reagents and DNA extraction card, allowing for sample-to-answer screening of single nucleotide polymorphisms (SNPs) typing of the CYP2C19 gene from untreated blood samples with minimal user operation. With all reagents and the DNA extraction card preloaded inside the capillary, this icLAMP system can achieve on-site pretreatment, extraction, amplification, and detection of nucleic acids within 150 min, without the requirement for advanced instruments. As icLAMP technology carries many advantages such as disposability, easy operation, low cost, and reduced cross contamination and biohazard risks, we expect this system to have a great impact on point-of-care (POC) nucleic acid detection. PMID:25242282

Zhang, Lu; Zhang, Yi; Wang, Chunyan; Feng, Qiang; Fan, Fei; Zhang, Guojun; Kang, Xixiong; Qin, Xuzhen; Sun, Jiashu; Li, Yinghui; Jiang, Xingyu

2014-10-21

369

Amino-Modified Tetraphenylethene Derivatives as Nucleic Acid Stain: Relationship between the Structure and Sensitivity.  

PubMed

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

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

2014-10-22

370

10/31/13 Chapters 7 & 19 -Nucleosides, Nucleotides and Nucleic Acids  

E-print Network

-methyl-G The free bases are hydrophobic and have low water solubility at pH 7. The exocyclic NH2 are non, nucleotides and nucleic acids function as vitamins and coenzymes, energy carriers, second messengers and the bases become more soluble. pKa's C N3 4.5 U N3 9.5 A N1 3.8 G N1 9.4 N7 2.4 #12;10/31/13 3

O'Neil, Joe

371

Hybrid Molecular Probe for Nucleic Acid Analysis in Biological Samples Chaoyong James Yang, Karen Martinez, Hui Lin, and Weihong Tan*  

E-print Network

Hybrid Molecular Probe for Nucleic Acid Analysis in Biological Samples Chaoyong James Yang, Karen Martinez, Hui Lin, and Weihong Tan* Center for Research at Bio/Nano Interface, Department of Chemistry

Tan, Weihong

372

Sequence-specific electrochemical detection of nucleic acids in real samples  

Microsoft Academic Search

This paper reviews past and current developments in the field of electrochemical biosensors with a focus on the sequence-specific\\u000a detection of nucleic acids in real samples. After electrochemical hybridization sensors had been first described in 1993,\\u000a it took nearly a decade until some of the many proposed protocols were indeed applied to real samples like blood or tissue.\\u000a Electrochemical transduction

Heiko Duwensee; Maren Mix; Gerd-Uwe Flechsig

2010-01-01

373

Nucleic acid stains as indicators of Giardia muris viability following cyst inactivation  

Microsoft Academic Search

A reliable viability assay for Giardia is required for the development of disinfection process design criteria and pathogen monitoring by water treatment utilities. Surveys of single-staining nucleic acid dyes (stain dead parasites only), and double-staining vital dye kits from Molecular Probes (stain live and dead parasites) were conducted to assess the viability of untreated, heat-killed, and chemically inactivated Giardia muris

R. Taghi-Kilani; L. L. Gyürék; P. J. Millard; G. R. Finch; M. Belosevic

1996-01-01

374

Electron microscopical localization of nucleic acids by means of nuclease-gold complexes  

Microsoft Academic Search

Summary  Nucleic acids can be specifically localized at the electron microscope level by means of enzyme-gold complexes. Two enzymes RNAase A and DNAase I were labelled with colloidal gold, and the enzyme-gold complexes obtained applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Using RNAase-gold, the rough endoplasmic reticulum and the nucleolus of different cells appeared densely labelled. With the DNAase-gold

Moise Bendayan

1981-01-01

375

Ebselen inhibits hepatitis C virus NS3 helicase binding to nucleic Acid and prevents viral replication.  

PubMed

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 ?M ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 ?M, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure-activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines. PMID:25126694

Mukherjee, Sourav; Weiner, Warren S; Schroeder, Chad E; Simpson, Denise S; Hanson, Alicia M; Sweeney, Noreena L; Marvin, Rachel K; Ndjomou, Jean; Kolli, Rajesh; Isailovic, Dragan; Schoenen, Frank J; Frick, David N

2014-10-17

376

1996 Oxford University Press 12791286Nucleic Acids Research, 1996, Vol. 24, No. 7 Incomplete factorial and response surface methods  

E-print Network

© 1996 Oxford University Press 1279­1286Nucleic Acids Research, 1996, Vol. 24, No. 7 Incomplete by stepwise multiple linear regression analysis. The concentrations of T7 RNA polymerase, DNA template, NTP

Yin, Y. Whitney

377

2-Acetylaminofluorene-modified probes for the indirect hybridocytochemical detection of specific nucleic acid sequences  

Microsoft Academic Search

A new approach is presented for the indirect hybridocytochemical localization of specific nucleic acid sequences in microscopic preparations. The method is based on the application of probes modified with N-acetoxy-2-acetylaminofluorene. After hybridization, the 2-acetylaminofluorene-labelled probes are recognized by antibodies directed against modified guanosine and visualized immunocytochemically. This procedure has been optimized on two model objects: mouse satellite DNA in interphase

J. E. Landegent; N. Jansen in de Wal; R. A. Baan; J. H. J. Hoeijmakers; Ploeg van der M

1984-01-01

378

MR contrast agent composed of cholesterol and peptide nucleic acids: Design, synthesis and cellular uptake  

Microsoft Academic Search

A new mRNA targeting contrast agent consisting of three main functional domains, (i) gadolinium based magnetic resonance reporter part, (ii) antisense peptide nucleic acids targeted to mRNA, and (iii) cholesterol as the delivery vector, was developed and synthesized. The new contrast agent showed efficient cellular uptake and significant contrast enhancement at very low labeling concentrations (0.5?M). However, after uptake into

Rajendra Joshi; Ritu Mishra; Rolf Pohmann; Jörn Engelmann

2010-01-01

379

A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands  

Microsoft Academic Search

In the preceding paper, a method to detect specific DNA sequences with mercurated nucleic acid probes and sulfhydryl-hapten ligands has been described. Due to the instability of the bond between mercury and a negatively charged sulfhydryl-hapten ligand (trinitrophenyl-glutathione), the in situ formed hybrid could not be detected. On basis of model system experiments it was suggested that this mercury-sulfhydryl bond

A. H. N. Hopman; J. Wiegant; P. Duijn

1986-01-01

380

A conformational study of nucleic acid phosphate ester bonds using phosphorus-31 nuclear magnetic resonance.  

PubMed Central

A systematic phosphorus-31 nuclear magnetic resonance study of some nucleic acid constituents (6-N-(dimethyl)adenylyl-(3',5')-uridine and some nucleotide methyl esters) is presented. The temperature dependent phosphorus-31 chemical shifts were analyzed by standard thermodynamic procedures. It is shown that gt conformations about the P-O ester bonds have a lower free energy content relative to gg conformers. PMID:440971

Haasnoot, C A; Altona, C

1979-01-01

381

Biologically relevant oxidants and terminology, classification and nomenclature of oxidatively generated damage to nucleobases and 2-deoxyribose in nucleic acids  

E-print Network

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 ...

Cadet, Jean

382

Performance of a commercial nucleic acid amplification test with extrapulmonary specimens for the diagnosis of tuberculosis  

Microsoft Academic Search

The laboratory diagnosis of tuberculosis (TB) on extrapulmonary specimens is particularly challenging. A number of commercial\\u000a nucleic acid amplification tests able to detect and identify Mycobacterium tuberculosis (MTB) complex directly from respiratory secretions have been developed, but their use on extrapulmonary samples still calls\\u000a for validation. The BDProbeTec ET Mycobacterium tuberculosis Complex Direct Detection Assay (DTB) was applied to 918

C. Piersimoni; S. Bornigia; G. Gherardi

383

Preventing disease transmission by deceased tissue donors by testing blood for viral nucleic acid  

Microsoft Academic Search

Nucleic acid testing (NAT) has reduced the risk of transmitting infectious disease through blood transfusion. Currently NAT\\u000a for HIV-1 and HCV are FDA licensed and performed by nearly all blood collection facilities, but HBV NAT is performed under\\u000a an investigational study protocol. Residual risk estimates indicate that NAT could potentially reduce disease transmission\\u000a through transplanted tissue. However, tissue donor samples

D. Michael Strong; Karen Nelson; Marge Pierce; Susan L. Stramer

2005-01-01

384

Kinetics and mechanism of the DNA double helix invasion by pseudocomplementary peptide nucleic acids  

Microsoft Academic Search

If adenines and thymines in two mutually complementary mixed-base peptide nucleic acid (PNA) oligomers are substituted with diaminopurines and thiouracils, respectively, so-called pseudocomplementary PNAs (pcPNAs) are created. Pairs of pcPNAs have recently demonstrated an ability to highly selectively target essentially any designated site on double-stranded DNA (dsDNA) by forming very stable PNA-DNA strand-displacement complexes via double duplex invasion (helix invasion).

Vadim V. Demidov; Ekaterina Protozanova; Konstantin I. Izvolsky; Christopher Price; Peter E. Nielsen; Maxim D. Frank-Kamenetskii

2002-01-01

385

Peptide nucleic acid (PNA) binding-mediated induction of human gamma- globin gene expression  

Microsoft Academic Search

Peptide nucleic acids (PNAs) can bind to homopurine\\/ homopyrimidine sequences of double-stranded DNA targets in a sequence-specific manner and form (PNA)2\\/DNA triplexes with single-stranded DNA D-loop structures at the PNA binding sites. These D-loop structures have been found to have a capacity to initiate transcription in vitro. If this strategy can be used to induce transcription of endogenous genes, it

Gan Wang; Xiaoxin Xu; Betty Pace; David A. Dean; Peter M. Glazer; Phillip Chan; Steven R. Goodman; Inna Shokolenko

1999-01-01

386

Rapid detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water using peptide nucleic acid probes  

Microsoft Academic Search

A new chemiluminescent in situ hybridization (CISH) method that provides simultaneous detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water within 1 working day has been developed. Individual micro-colonies of P. aeruginosa were detected directly on membrane filters following 5 h of growth by use of soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeted to a species-specific sequence in

Henrik Stender; Adam Broomer; Kenneth Oliveira; Heather Perry-O’Keefe; Jens J Hyldig-Nielsen; Andrew Sage; Barbara Young; James Coull

2000-01-01

387

In vivo hybridization of technetium-99m-labeled peptide nucleic acid (PNA)  

Microsoft Academic Search

Hybridization of a radiolabeled single-stranded DNA oligonucleotide with its single-stranded complement in vivo has not yet been convincingly demonstrated. A contributing factor may be unfavorable in vivo properties of the phosphodiester and phosphorothioate DNAs. Peptide nucleic acid (PNA) oligomers have been reported to possess in vivo properties more suitable for radiopharmaceutical applications. We have radiolabeled an amine-derivatized 15-base PNA oligomer

G. Mardirossian; K. Lei; M. Rusckowski

1997-01-01

388

Sequence-Specific Targeting of Duplex DNA by Peptide Nucleic Acids via Triplex Strand Invasion  

Microsoft Academic Search

Because of a set of exceptional chemical, physical, and biological properties, polyamide or peptide nucleic acids (PNAs) hold a distinctive position among various synthetic ligands designed for DNA-targeting purposes. Cationic pyrimidine PNAs (cpyPNAs) represent a special group of PNAs, which effectively form strand invasion triplexes with double-stranded DNA (dsDNA) also known as P-loops. Extraordinary stability of the invasion triplexes and

Vadim V. Demidov; Maxim D. Frank-Kamenetskii

2001-01-01

389

Peptide Nucleic Acid-Targeted Mutagenesis of a Chromosomal Gene in Mouse Cells  

Microsoft Academic Search

Peptide nucleic acids (PNAs) can bind to single-stranded DNA by Watson-Crick base pairing and can form triple helices via Hoogsteen bonding to DNA\\/PNA duplexes. A single dimeric PNA molecule can form a clamp via both double- and triple-helix formation. We designed PNAs to bind as clamps to a site in the supFG1 mutation reporter gene carried within a chromosomally integrated,

A. Fawad Faruqi; Michael Egholm; Peter M. Glazer

1998-01-01

390

Covalent functionalization of single walled carbon nanotubes with peptide nucleic acid: Nanocomponents for molecular level electronics  

Microsoft Academic Search

Imparting molecular recognition to carbon nanotubes (CNTs) by conjugating them with bio-molecules has been an area of great interest as the resulting highly functionalized CNT-bioconjugates find their applications in various fields like molecular level electronics, pharmaceuticals, drug delivery, novel materials and many others. In this work we demonstrate the synthesis of functionally engineered single walled carbon nanotubes (SWNTs)-peptide nucleic acid

Krishna V. Singh; Rajeev R. Pandey; Xu Wang; Roger Lake; Cengiz S. Ozkan; Kang Wang; Mihrimah Ozkan

2006-01-01

391

Peptide Nucleic Acids as Tools for Single-Molecule Sequence Detection and Manipulation  

NASA Astrophysics Data System (ADS)

The ability to strongly and sequence-specifically attach modifications such as fluorophores and haptens to individual double-stranded (ds) DNA molecules is critical to a variety of single-molecule experiments. We propose using modified peptide nucleic acids (PNAs) for this purpose and implement them in two model single-molecule experiments where individual DNA molecules are manipulated via microfluidic flow and optical tweezers, respectively. We demonstrate that PNAs are versatile and robust sequence-specific tethers.

Zohar, Hagar; Hetherington, Craig; Bustamante, Carlos; Muller, Susan

2011-03-01

392

In Vitro Transcriptional and Translational Block of the bcl-2 Gene Operated by Peptide Nucleic Acid  

Microsoft Academic Search

The antisense and antigene activity of peptide nucleic acid (PNA) targeted to the human B-cell lymphoma (bcl)-2 gene was evaluated in vitro. Several PNAs complementary to different sequences of bcl-2, including the start codon and the 5?-untranslated region (5?-UTR), were tested. One PNA directed against the AUG start codon and another recognizing the 5?-UTR were able to specifically reduce Bcl-2

Luca Mologni; Peter E. Nielsen; Carlo Gambacorti-Passerini

1999-01-01

393

Down-regulation of amyloid precursor protein by peptide nucleic acid in vivo  

Microsoft Academic Search

Alzheimer’s disease (AD) is a neurodegenerative disease associated with increased expression of amyloid precursor protein\\u000a (APP) and the deposition of its proteolytic cleavage products, the amyloid-? peptides, A?1–40 and A?1–42. Peptide nucleic acids (PNAs) have been shown to block the expression of proteins at transcriptional and translational levels.\\u000a In this study we used a sense and an antisense PNA specifically

Mona Boules; Katrina Williams; Elisa Gollatz; Abdul Fauq; Elliott Richelson

2004-01-01

394

Single-stranded nucleic acid recognition: is there a code after all?  

PubMed

Proteins that bind single-stranded nucleic acids have crucial roles in cells, and structural analyses have contributed to a better understanding of their functions. In this issue of Structure, Dickey and colleagues describe several high resolution structures of a single OB-fold bound to different single-stranded DNA (ssDNA) sequences and reveal a spectacular co-adaptability of the protein/ssDNA interactions. PMID:23312030

Cléry, Antoine; Boudet, Julien; Allain, Frédéric H-T

2013-01-01

395

Nucleic Acid Structure Analysis: A Users Guide to a Collection of New Analysis Programs  

Microsoft Academic Search

Common nomenclature describing the geometry of nucleic acid structures was established at a 1988 EMBO Workshop on DNA Curvature and Bending (Diekmann, S. (1988) J. Mol. Biol. 208, 787–791; Diekmann, S. (1989) The EMBO Journal 8, 1–4; Sarma, RH. (1988) J. Biomol. Structure & Dynamics 6, 391–395; Dickerson, R.E. (1989) J. Biomol. Structured Dynamics 6, 627–634; Dickerson, RE. et al.

Maria S. Babcock; Edwin P. D. Pednault; Wilma K. Olson

1993-01-01

396

Comparison of Automated and Manual Nucleic Acid Extraction Methods for Detection of Enterovirus RNA  

Microsoft Academic Search

Automated nucleic acid extraction is an attractive alternative to labor-intensive manual methods. We compared two automated methods, the BioRobot M48 instrument (Qiagen, Inc.) and MagNA Pure (Roche Applied Sciences) methods, to two manual methods, the QIAamp Viral RNA Mini kit (Qiagen) and TRIzol (Invitrogen), for the extraction of enterovirus RNA. Analytical sensitivity was assessed by dilution analysis of poliovirus type

Julia H. Knepp; Melissa A. Geahr; Michael S. Forman; Alexandra Valsamakis

2003-01-01

397

MACROMOLECULES, Part 1* Carbohydrates, Lipids, and Nucleic Acids  

E-print Network

of functions in organisms. For example: · they are major constituents of cellular membranes · they are the most about what it means when we say a compound is "reduced" or oxidized when we consider cellular respiration (so-called "catabolism" and "anabolism"). #12;unsaturated. A saturated fatty acid contains

Prestwich, Ken

398

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

E-print Network

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 are in accordance with the available experimental data, and through systematic calculations, we obtained the empirical formulas for lp as a function of Na, Mg and chain length.

Feng-Hua Wang; Yuan-Yan Wu; Zhi-Jie Tan

2012-11-23

399

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

PubMed Central

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. PMID:24193511

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

2013-01-01

400

Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain.  

PubMed

RNA interference is a conserved mechanism that regulates gene expression in response to the presence of double-stranded (ds)RNAs. The RNase III-like enzyme Dicer first cleaves dsRNA into 21-23-nucleotide small interfering RNAs (siRNAs). In the effector step, the multimeric RNA-induced silencing complex (RISC) identifies messenger RNAs homologous to the siRNAs and promotes their degradation. The Argonaute 2 protein (Ago2) is a critical component of RISC. Both Argonaute and Dicer family proteins contain a common PAZ domain whose function is unknown. Here we present the three-dimensional nuclear magnetic resonance structure of the Drosophila melanogaster Ago2 PAZ domain. This domain adopts a nucleic-acid-binding fold that is stabilized by conserved hydrophobic residues. The nucleic-acid-binding patch is located in a cleft between the surface of a central beta-barrel and a conserved module comprising strands beta3, beta4 and helix alpha3. Because critical structural residues and the binding surface are conserved, we suggest that PAZ domains in all members of the Argonaute and Dicer families adopt a similar fold with nucleic-acid binding function, and that this plays an important part in gene silencing. PMID:14615801

Lingel, Andreas; Simon, Bernd; Izaurralde, Elisa; Sattler, Michael

2003-11-27

401

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

PubMed Central

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

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

2013-01-01

402

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

PubMed

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

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

2013-10-21

403

A nanocarrier system for the delivery of nucleic acids targeted to a pancreatic beta cell line.  

PubMed

Pancreatic ? cells secrete insulin in response to glucose levels and thus are involved in controlling blood glucose levels. A line of pancreatic ? cells "MIN6" has been used in studies related to the function of ? cells and diabetes therapy. Regulating gene expression in MIN6 cells could accelerate these studies, but an efficient method for the transfection of nucleic acids targeted to MIN6 cells is required. We report here on a liposome-based carrier targeted to pancreatic ? cells (Multifunctional envelope-type nano device for pancreatic ? cells, ?-MEND). We identified a lipid composition for use in preparing the ?-MEND, which permits the particles to be efficiently internalized into MIN6, as evidenced by flow cytometry analyses. Intracellular observation by confocal laser scanning microscopy showed that the ?-MEND efficiently delivered the oligo nucleic acids to the cytosol of MIN6 cells. Moreover, using a ?-MEND encapsulating a 2'-O-Methyl RNA complementary to a microRNA that suppresses insulin secretion, the knockdown of the targeted microRNA and an up-regulation of insulin secretion were observed in MIN6. Thus, the ?-MEND holds promise as an efficient system for delivering nucleic acids targeted to MIN6 and can contribute to research and therapy aimed at diabetes. PMID:24816283

Yamada, Yuma; Tabata, Mai; Yasuzaki, Yukari; Nomura, Masatoshi; Shibata, Atsushi; Ibayashi, Yuta; Taniguchi, Yosuke; Sasaki, Shigeki; Harashima, Hideyoshi

2014-08-01

404

Maximizing the electromagnetic and chemical resonances of surface-enhanced Raman scattering for nucleic acids.  

PubMed

Although surface-enhanced Raman spectroscopy (SERS) has previously been performed with nucleic acids, the measured intensities for each nucleic acid have varied significantly depending on the SERS substrate and excitation wavelength. We have demonstrated that the charge-transfer (CT) mechanism, also known as the chemical enhancement of SERS, is responsible for the discrepancies previously reported in literature. The electronic states of cytosine and guanine attached to silver atoms are computationally calculated and experimentally measured to be in the visible range, which leads to a resonance Raman effect at the corresponding maximum wavelengths. The resulting SERS measurements are in good agreement with the simulated values, in which cytosine-silver shows stronger enhancement at 532 nm and guanine-silver shows stronger enhancement at 785 nm. An atomic layer of aluminum oxide is deposited on substrates to prevent charge-transfer, and corresponding measurements show weaker Raman signals caused by the suppression of the chemical resonance. These findings suggest the optimal SERS signal can be achieved by tuning the excitation wavelength to match both the electromagnetic and chemical resonances, paving the way for future single molecule detection of nucleic acids other than adenine. PMID:25065837

Freeman, Lindsay M; Pang, Lin; Fainman, Yeshaiahu

2014-08-26

405

Mass spectrometry-based identification of proteins interacting with nucleic acids.  

PubMed

The identification of the regulatory proteins that control DNA transcription as well as RNA stability and translation represents a key step in the comprehension of gene expression regulation. Those proteins can be purified by DNA- or RNA-affinity chromatography, followed by identification by mass spectrometry. Although very simple in the concept, this represents a real technological challenge due to the low abundance of regulatory proteins compared to the highly abundant proteins binding to nucleic acids in a nonsequence-specific manner. Here we review the different strategies that have been set up to reach this purpose, discussing the key parameters that should be considered to increase the chances of success. Typically, two categories of biological questions can be distinguished: the identification of proteins that specifically interact with a precisely defined binding site, mostly addressed by quantitative mass spectrometry, and the identification in a non-comparative manner of the protein complexes recruited by a poorly characterized long regulatory region of nucleic acids. Finally, beside the numerous studies devoted to in vitro-assembled nucleic acid-protein complexes, the scarce data reported on proteomic analyses of in vivo-assembled complexes are described, with a special emphasis on the associated challenges. PMID:24060998

Tacheny, A; Dieu, M; Arnould, T; Renard, P

2013-12-01

406

Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification.  

PubMed

A simple isothermal nucleic-acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60 degrees C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or non-specific amplification. Instead, 'primers' are generated and accumulated during the reaction. The circular probe carries only two sequences: (i) a hybridization sequence to the sample DNA and (ii) a recognition sequence of the nicking enzyme. In PG-RCA, the circular probe first hybridizes with the sample DNA, and then a cascade reaction of linear rolling circle amplification and nicking reactions takes place. In contrast with conventional linear rolling circle amplification, the signal amplification is in an exponential mode since many copies of 'primers' are successively produced by multiple nicking reactions. Under the optimized condition, we obtained a remarkable sensitivity of 84.5 ymol (50.7 molecules) of synthetic sample DNA and 0.163 pg (approximately 60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG-RCA to various molecular diagnostic assays. PMID:19106144

Murakami, Taku; Sumaoka, Jun; Komiyama, Makoto

2009-02-01

407

An injection molded microchip for nucleic acid purification from 25 microliter samples using isotachophoresis.  

PubMed

We present a novel microchip device for purification of nucleic acids from 25?L biological samples using isotachophoresis (ITP). The device design incorporates a custom capillary barrier structure to facilitate robust sample loading. The chip uses a 2mm channel width and 0.15mm depth to reduce processing time, mitigate Joule heating, and achieve high extraction efficiency. To reduce pH changes in the device due to electrolysis, we incorporated a buffering reservoir physically separated from the sample output reservoir. To reduce dispersion of the ITP-focused zone, we used optimized turn geometries. The chip was fabricated by injection molding PMMA and COC plastics through a commercial microfluidic foundry. The extraction efficiency of nucleic acids from the device was measured using fluorescent quantification, and an average recovery efficiency of 81% was achieved for nucleic acid masses between 250pg and 250ng. The devices were also used to purify DNA from whole blood, and the extracted DNA was amplified using qPCR to show the PCR compatibility of the purified sample. PMID:24485540

Marshall, L A; Rogacs, A; Meinhart, C D; Santiago, J G

2014-02-28

408

FRETmatrix: a general methodology for the simulation and analysis of FRET in nucleic acids  

PubMed Central

Förster resonance energy transfer (FRET) is a technique commonly used to unravel the structure and conformational changes of biomolecules being vital for all living organisms. Typically, FRET is performed using dyes attached externally to nucleic acids through a linker that complicates quantitative interpretation of experiments because of dye diffusion and reorientation. Here, we report a versatile, general methodology for the simulation and analysis of FRET in nucleic acids, and demonstrate its particular power for modelling FRET between probes possessing limited diffusional and rotational freedom, such as our recently developed nucleobase analogue FRET pairs (base–base FRET). These probes are positioned inside the DNA/RNA structures as a replacement for one of the natural bases, thus, providing unique control of their position and orientation and the advantage of reporting from inside sites of interest. In demonstration studies, not requiring molecular dynamics modelling, we obtain previously inaccessible insight into the orientation and nanosecond dynamics of the bases inside double-stranded DNA, and we reconstruct high resolution 3D structures of kinked DNA. The reported methodology is accompanied by a freely available software package, FRETmatrix, for the design and analysis of FRET in nucleic acid containing systems. PMID:22977181

Preus, S?ren; Kilsa, Kristine; Miannay, Francois-Alexandre; Albinsson, Bo; Wilhelmsson, L. Marcus

2013-01-01

409

Evaluation of NucliSens easyMAG for Automated Nucleic Acid Extraction from Various Clinical Specimens  

Microsoft Academic Search

The objectives of this study were to evaluate the performance of the NucliSens easyMAG platform for nucleic acid extraction from different clinical specimens compared to NucliSens miniMAG platform and manual QIAGEN extraction. The NucliSens easyMAG and the NucliSens miniMAG showed equal performance on 215 throat swabs since real-time nucleic acid sequence-based amplification scored the same samples positive for Mycoplasma pneumoniae

K. Loens; K. Bergs; D. Ursi; H. Goossens; M. Ieven

2007-01-01

410

Synthesis of a Stable and Specific Surface Plasmon Resonance Biosensor Surface Employing Covalently Immobilized Peptide Nucleic Acids  

Microsoft Academic Search

Biosensors allow the real-time and label-free observation of biochemical reactions between various ligands including antigen-antibody reactions and nucleic acids hybridizations. In our studies, we used a surface plasmon resonance biosensor to elucidate the hybridization characteristics of a peptide nucleic acid (PNA) ligand immobilized on sensor surfaces either through covalent or streptavidin- biotin coupling. A biotin-labeled PNA was employed in the

Martin Burgener; Michael Sanger; Urs Candrian

2000-01-01

411

Fluorescent polymeric transducer for the rapid, simple, and specific detection of nucleic acids at the zeptomole level.  

PubMed

We report the specific detection of a few hundred molecules of genetic material using a fluorescent polythiophene biosensor. Such recognition is based on simple electrostatic interactions between a cationic polymeric optical transducer and the negatively charged nucleic acid target and can be done in less than 1 h, simply and affordably, and without any chemical reaction. This simple system is versatile enough to detect nucleic acids of various lengths, including a segment from the RNA genome of the Influenza virus. PMID:15053613

Doré, Kim; Dubus, Sébastien; Ho, Hoang-Anh; Lévesque, Isabelle; Brunette, Maryse; Corbeil, Geneviève; Boissinot, Maurice; Boivin, Guy; Bergeron, Michel G; Boudreau, Denis; Leclerc, Mario

2004-04-01

412

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

NASA Astrophysics Data System (ADS)

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 duplexes alone interacted with cations distinct from a specific sequence. Under physiologically relevant conditions, a duplex of RNA polyguanine-polycitidine was highly responsive and able to sequester cations to the middle of the purine stretches. The least responsive structure was a DNA polyadenine-polythymine duplex. A random sequence DNA duplex contorted into an RNA-like helix resulted in cationic dynamics similar to RNA systems. These studies showed that cation diffusive binding events in nucleic acid duplex structures are sequence specific and heavily influenced by structural aspects helical forms to account for much of the differences observed. Although structural information in nucleic acids is encoded within their sequence, linking amino acid sequence to protein structure is murkier; the structural information within proteins is encoded by the folding process itself: a complex phenomenon driven toward the equilibrium state of the active conformation. Upwards of two thirds of a protein's sequence can be substituted with similar amino acids without significantly perturbing its function; conserved residues of about 10% seem to be vital; since evolutionary selection pressure in proteins operates 3-dimenionally, a linear sequence is partially informative. We explored this problem by folding de-novo the cytosolic portion of the membrane protein, cellulose synthase, CESA1 from upland cotton, Gossypium hirsutum (Ghcesa1). The cytoplasmic region was generated by homology modeling and refined with molecular dynamics. These mutations impair local structural flexibility which likely results in cellulose that is produced at a lower rate and is less crystalline. Additional modeling of fragments of cellulose synthases from the model plant, Arabidopsis thaliana, offered novel insights into the function of conserved cytosolic domains within plant cellulose synthases. Transport mechanisms related to the transmembrane region revealed significant differences between plants and a bacterial complex. These studies generated possible mutations that may allow for the creation of

Sethaphong, Latsavongsakda

413

Magnetite nanoparticle with positively charged surface for immobilization of peptide nucleic acid and deoxyribonucleic acid.  

PubMed

We herein report the surface modification of magnetite nanoparticle (MNP) with the (co)polymer of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and/or diethylamino ethyl methacrylate (DEAEMA) via atom transfer radical polymerization (ATRP) for use as anion exchanger solid support for detection of DNA sequence using peptide nucleic acid (PNA) probe. Molar ratio of the PEGMA:DEAEMA (co)polymer was systematically varied to tune the positive charges on the particle surface. Kinetic studies of the (co)polymerizations were investigated via 1HNMR to disclose the relative reactivity of the (co)polymers in the reaction. Zeta potential of the (co)polymer-grafted MNP was analyzed by photo correlation spectroscopy (PCS). Transmission electron microscopy (TEM) and PCS indicated an improvement in the particle dispersibility in water upon quaternization of the DEAEMA entities grafted on the particle surface. From the preliminary results, these (co)polymer-grafted MNPs can be used as a nanosolid support to differentiate between full match and single-base mismatch DNA sequences using an acpcPNA probe. These novel cationic MNPs might be efficiently applicable for use as a magnetically guidable tool for detection of DNA sequences. PMID:23980499

Theppaleak, T; Rutnakornpituk, B; Wichai, U; Vilaivan, T; Rutnakornpituk, M

2013-09-01

414

The biology of FTO: from nucleic acid demethylase to amino acid sensor.  

PubMed

Genome-wide association studies have revealed that single-nucleotide polymorphisms in the first intron of the gene encoding fat mass and obesity-associated protein (FTO) are robustly associated with BMI and obesity. Subsequently, this association with body weight, which is replicable across multiple populations and different age groups, has been unequivocally linked to increased food intake. Although evidence from a number of animal models with perturbed FTO expression indicates a role for FTO in energy homeostasis, to date, no conclusive link has been made between the risk alleles and FTO expression or its physiological role. FTO is a nucleic acid demethylase, and a deficiency in FTO leads to a complex phenotype highlighted by postnatal growth retardation, pointing to some fundamental developmental role. Recent emerging data now points to a role for FTO in the sensing of nutrients and the regulation of translation and growth. In this review, we explore the in vivo and in vitro evidence detailing the complex biology of FTO and discuss how these might link to the regulation of body weight. PMID:23896822

Gulati, Pawan; Yeo, Giles S H

2013-10-01

415

Backbone-base inclination as a fundamental determinant of nucleic acid self- and cross-pairing  

PubMed Central

The crystal structure of the duplex formed by oligo(2?,3?-dideoxy-?-d-glucopyranosyl)nucleotides (homo-DNA) revealed strongly inclined backbone and base-pair axes [Egli,M., Pallan,P.S., Pattanayek,R., Wilds,C.J., Lubini,P., Minasov,G., Dobler,M., Leumann,C.J. and Eschenmoser,A. (2006) Crystal structure of homo-DNA and nature's choice of pentose over hexose in the genetic system. J. Am. Chem. Soc., 128, 10847–10856]. This inclination is easily perceived because homo-DNA exhibits only a modest helical twist. Conversely, the tight coiling of strands conceals that the backbone-base inclinations for A- (DNA and RNA) and B-form (DNA) duplexes differ considerably. We have defined a parameter ?B that corresponds to the local inclination between sugar-phosphate backbone and base plane in nucleic acid strands. Here, we show its biological significance as a predictive measure for the relative strand polarities (antiparallel, aps, or parallel, ps) in duplexes of DNA, RNA and artificial nucleic acid pairing systems. The potential of formation of ps duplexes between complementary 16-mers with eight A and U(T) residues each was investigated with DNA, RNA, 2?-O-methylated RNA, homo-DNA and p-RNA, the ribopyranosyl isomer of RNA. The thermodynamic stabilities of the corresponding aps duplexes were also measured. As shown previously, DNA is capable of forming both ps and aps duplexes. However, all other tested systems are unable to form stable ps duplexes with reverse Watson–Crick (rWC) base pairs. This observation illustrates the handicap encountered by nucleic acid systems with inclinations ?B that differ significantly from 0° to form a ps rWC paired duplex. Accordingly, RNA with a backbone-base inclination of ?30°, pairs strictly in an aps fashion. On the other hand, the more or less perpendicular orientation of backbone and bases in DNA allows it to adopt a ps rWC paired duplex. In addition to providing a rationalization of relative strand polarity with nucleic acids, the backbone-base inclination parameter is also a determinant of cross-pairing. Thus, systems with strongly deviating ?B angles will not pair with each other. Nucleic acid pairing systems with significant backbone-base inclinations can also be expected to display different stabilities depending on which terminus carries unpaired nucleotides. The negative inclination of RNA is consistent with the higher stability of duplexes with 3?- compared to those with 5?-dangling ends. PMID:17905816

Pallan, Pradeep S.; Lubini, Paolo; Bolli, Martin; Egli, Martin

2007-01-01

416

[Effect of parenteral nutrition on the nucleic acid content in normal rat tissues and in thyrotoxicosis].  

PubMed

The content of nucleic acids in tissues of healthy animals and those suffering from thyrotoxicosis was studied as affected by parenteral administration of amino acid mixture of moriamine S-2 and casein hydrolysate. The content of RNA in the skeletal muscles, heart and liver is established to change considerably under the effect of nitrogenous media. With administration of moriamine S-2 or caseine hydrolysate the higher level of RNA in tissues with thyrotoxicosis, is normalized, especially in the skeletal muscles. The character of changes depends essentially on properties and composition of the administered preparations. PMID:411201

Hlanz, R M; Skovrons'ka, E V; Vovk, H P

1977-01-01

417

Solution Preserves Nucleic Acids in Body-Fluid Specimens  

NASA Technical Reports Server (NTRS)

A solution has been formulated to preserve deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in specimens of blood, saliva, and other bodily fluids. Specimens of this type are collected for diagnostic molecular pathology, which is becoming the method of choice for diagnosis of many diseases. The solution makes it possible to store such specimens at room temperature, without risk of decomposition, for subsequent analysis in a laboratory that could be remote from the sampling location. Thus, the solution could be a means to bring the benefits of diagnostic molecular pathology to geographic regions where refrigeration equipment and diagnostic laboratories are not available. The table lists the ingredients of the solution. The functions of the ingredients are the following: EDTA chelates divalent cations that are necessary cofactors for nuclease activity. In so doing, it functionally removes these cations and thereby retards the action of nucleases. EDTA also stabilizes the DNA helix. Tris serves as a buffering agent, which is needed because minor contaminants in an unbuffered solution can exert pronounced effects on pH and thereby cause spontaneous degradation of DNA. SDS is an ionic detergent that inhibits ribonuclease activity. SDS has been used in some lysis buffers and as a storage buffer for RNA after purification. The use of the solution is straightforward. For example, a sample of saliva is collected by placing a cotton roll around in the subject's mouth until it becomes saturated, then the cotton is placed in a collection tube. Next, 1.5 mL of the solution are injected directly into the cotton and the tube is capped for storage at room temperature. The effectiveness of the solution has been demonstrated in tests on specimens of saliva containing herpes simplex virus. In the tests, the viral DNA, as amplified by polymerase chain reaction, was detected even after storage for 120 days.

Pierson, Duane L.; Stowe, Raymond P.

2004-01-01

418

Nucleic acid-based differential diagnostic assays for feline coronavirus.  

PubMed

Feline coronavirus (FCoV) is a pleomorphic, enveloped, positive-sense single-stranded RNA virus. Owing to the differences in its genotype, FCoV belongs to a separate clade along with other viruses, such as transmissible gastroenteritis virus (TGEV) and canine coronavirus (CCoV), which can be isolated from cats. In this study, a PCR assay was developed to differentiate these coronaviruses concurrently. Multiplex differential RT-PCR was performed with primers based on the highly conserved coronavirus membrane protein. Three primer sets were designed: a primer pair (S1 and S2) that can bind to conserved sequences in all target coronaviruses, a CCoV-specific primer (S3), and a TGEV-specific primer (S4). Because of the high sequence homology among FCoV, CCoV, and TGEV, a nucleotide preceding the last pair of dissimilar nucleotides in S3 and S4 was substituted with an inosine to allow primer binding. This assay could detect and differentiate FCoV (n=7), CCoV (n=4), and TGEV (n=8) precisely and did not show any cross-reactivity with other pathogens. These results suggest that this molecular approach provides a rapid and reliable way to detect FCoV, especially in feline clinical specimens. PMID:25088974

Han, Jae-Ik; Kang, Shien-Young; Yoon, Kyoung-Jin; Na, Ki-Jeong

2014-11-01

419

Di-heterometalation of thiol-functionalized peptide nucleic acids  

PubMed Central

As a proof-of-principle, two hetero-bimetallic PNA oligomers containing a ruthenium(II) polypyridyl and a cyclopentadienyl manganese tricarbonyl complex have been prepared by serial combination of solid-phase peptide coupling and in-solution thiol chemistry. Solid-phase N-terminus attachment of Ru(II)-polypyridyl carboxylic acid derivative, C1, onto the thiol-functionalized PNA backbone (H-a-a-g-t-c-t-g-c-linker-cys-NH2) has been performed by standard peptide coupling method. As two parallel approaches, the strong affinity of thiols for maleimide and haloacetyl group has been exploited for subsequent post-SPPS addition of cymantrene-based organometallic cores, C2 and C3. Michael-like addition and thioether ligation of thiol functionalized PNA1 (H-gly-a-a-g-t-c-t-g-c-linker-cys-NH2) and PNA2 (C1-a-a-g-t-c-t-g-c-linker-cys-NH2) to cymantrene maleimide and chloroacetyl derivatives, C2 and C3, respectively, has been performed. The synthesized ruthenium(II)-cymantrenyl PNA oligomers have been characterized by mass spectrometry (ESI-MS) and IR spectroscopy. The distinct Mn-CO vibrational IR stretches, between 1,924–2,074 cm?1, have been used as markers to confirm the presence of cymantrenyl units in the PNA sequences and the purity of the HPLC-purified PNA thioethers assessed using LC-MS. PMID:23422249

Joshi, Tanmaya; Patra, Malay; Spiccia, Leone; Gasser, Gilles

2013-01-01

420

Structural transformation induced by locked nucleic acid or 2?-O-methyl nucleic acid site-specific modifications on thrombin binding aptamer  

PubMed Central

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, which is frequently modified to increase its analytical performance. Results The experimental results showed that when two or more nucleotides were substituted with LNA or OMeNA, the anti-parallel TBA structure was transformed into an unstructured random conformation in a 50 mM K+ environment; OMeNA appeared to have greater power to induce this transformation. However, the native TBA was unstructured in a 50 mM Ca2+ environment, whereas four or more LNA- or OMeNA- substitutions could convert this unstructured TBA into a parallel quadruplex structure. PAGE mobility measurements suggested that these TBAs might be a dimeric form. Conclusion LNA or 2'-OMeNA site-specific modifications induced G-quadruplex structural transformation of TBA, which enriched our understanding of the intrinsic G-quadrupex forming property and affinity of LNA and OMeNA modifications. This study demonstrates possible applications in the regulation of gene expression (i.e. manual intervention of gene therapy), genetic analyses, molecular diagnosis and the construction of nano-scale biostructures. PMID:24642032

2014-01-01

421

Application of peptide nucleic acid to the direct detection of deoxyribonucleic acid amplified by polymerase chain reaction  

Microsoft Academic Search

Double-stranded DNA amplified by polymerase chain reaction (PCR) was detected by peptide nucleic acid (PNA) using a BIAcore™ 2000 biosensor based on surface plasmon resonance (SPR). PNA is an artificial oligo amide that is capable of forming highly stable complexes with complementary oligonucleotides. We succeeded in the direct detection of double-stranded DNA, amplified by PCR with high-sequence specificity. It was

Shinya Sawata; Eriko Kai; Kazunori Ikebukuro; Tetsuya Iida; Takeshi Honda; Isao Karube

1999-01-01

422

NEATTILL: A simplified procedure for nucleic acid extraction from arrayed tissue for TILLING and other high-throughput reverse genetic applications  

PubMed Central

Background TILLING (Targeting Induced Local Lesions in Genomes) is a reverse genetics procedure for identifying point mutations in selected gene(s) amplified from a mutagenized population using high-throughput detection platforms such as slab gel electrophoresis, capillary electrophoresis or dHPLC. One essential pre-requisite for TILLING is genomic DNA isolation from a large population for PCR amplification of selected target genes. It also requires multiplexing of genomic DNA isolated from different individuals (pooling) in typically 8-fold pools, for mutation scanning, and to minimize the number of PCR amplifications, which is a strenuous and long-drawn-out work. We describe here a simplified procedure of multiplexing, NEATTILL (Nucleic acid Extraction from Arrayed Tissue for TILLING), which is rapid and equally efficient in assisting mutation detection. Results The NEATTILL procedure was evaluated for the tomato TILLING platform and was found to be simpler and more efficient than previously available methods. The procedure consisted of pooling tissue samples, instead of nucleic acid, from individual plants in 96-well plates, followed by DNA isolation from the arrayed samples by a novel protocol. The three variants of the NEATTILL procedure (vast, in-depth and intermediate) can be applied across various genomes depending upon the population size of the TILLING platform. The 2-D pooling ensures the precise confirmation of the coordinates of the positive mutant line while scanning complementary plates. Choice of tissue for arraying and nucleic acid isolation is discussed in detail with reference to tomato. Conclusion NEATTILL is a convenient procedure that can be applied to all organisms, the genomes of which have been mutagenized and are being scanned for multiple alleles of various genes by TILLING for understanding gene-to-phenotype relationships. It is a time-saving, less labour intensive and reasonably cost-effective method. Tissue arraying can cut costs by up to 90% and minimizes the risk of exposing the DNA to nucleases. Before arraying, different tissues should be evaluated for DNA quality, as the case study in tomato showed that cotyledons rather than leaves are better suited for DNA isolation. The protocol described here for nucleic acid isolation can be generally adapted for large-scale projects such as insertional mutagenesis, transgenic confirmation, mapping and fingerprinting which require isolation of DNA from large populations. PMID:20181012

2010-01-01

423

Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1  

PubMed Central

The mechanisms by which tumor microenvironments modulate nucleic acid–mediated innate immunity remain unknown. Here we identify the receptor TIM-3 as key in circumventing the stimulatory effects of nucleic acids in tumor immunity. Tumor-associated dendritic cells (DCs) in mouse tumors and patients with cancer had high expression of TIM-3. DC-derived TIM-3 suppressed innate immune responses through the recognition of nucleic acids by Toll-like receptors and cytosolic sensors via a galectin-9-independent mechanism. In contrast, TIM-3 interacted with the alarmin HMGB1 to interfere with the recruitment of nucleic acids into DC endosomes and attenuated the therapeutic efficacy of DNA vaccination and chemotherapy by diminishing the immunogenicity of nucleic acids released from dying tumor cells. Our findings define a mechanism whereby tumor microenvironments suppress antitumor immunity mediated by nucleic acids. PMID:22842346

Chiba, Shigeki; Baghdadi, Muhammad; Akiba, Hisaya; Yoshiyama, Hironori; Kinoshita, Ichiro; Dosaka-Akita, Hirotoshi; Fujioka, Yoichiro; Ohba, Yusuke; Gorman, Jacob V; Colgan, John D; Hirashima, Mitsuomi; Uede, Toshimitsu; Takaoka, Akinori; Yagita, Hideo; Jinushi, Masahisa

2013-01-01

424

Control of the photocatalytic activity of bimetallic complexes of pyropheophorbide-a by nucleic acids.  

PubMed

Photocatalytic activity of a photosensitizer (PS) in an oligodeoxyribonucleotide duplex 5'-PS~ODN1/ODN2~Q-3' is inhibited because of close proximity of a quencher Q. The ODN2 in this duplex is selected to be longer than the ODN1. Therefore, in the presence of a nucleic acid (analyte), which is fully complementary to the ODN2 strand, the duplex is decomposed with formation of an analyte/ODN2~Q duplex and a catalytically active, single stranded PS~ODN1. In this way the catalytic activity of the PS can be controlled by the specific nucleic acids. We applied this reaction earlier for the amplified detection of ribonucleic acids in live cells (Arian, D.; Cló, E.; Gothelf, K.; Mokhir, A. Chem.-Eur. J.2010, 16(1), 288). As a photosensitizer (PS) we used In(3+)(pyropheophorbide-a)chloride and as a quencher (Q)--Black-Hole-Quencher-3 (BHQ-3). The In(3+) complex is a highly active photocatalyst in aqueous solution. However, it can coordinate additional ligands containing thiols (e.g., proteins, peptides, and aminoacids), that modulate properties of the complex itself and of the corresponding bio- molecules. These possible interactions can lead to undesired side effects of nucleic acid controlled photocatalysts (PS~ODN1/ODN2?Q) in live cells. In this work we explored the possibility to substitute the In(3+) complex for those ones of divalent metal ions, Zn(2+) and Pd(2+), which exhibit lower or no tendency to coordinate the fifth ligand. We found that one of the compounds tested (Pd(pyropheophorbide-a) is as potent and as stable photosensitizer as its In(3+) analogue, but does not coordinate additional ligands that makes it more suitable for cellular applications. When the Pd complex was introduced in the duplex PS~ODN1/ODN2~Q as a PS, its photocatalytic activity could be controlled by nucleic acids as efficiently as that of the corresponding In(3+) complex. PMID:22047611

Arian, Dumitru; Kovbasyuk, Larisa; Mokhir, Andriy

2011-12-01

425

Insights into peptide nucleic acid (PNA) structural features: The crystal structure of a D-lysine-based chiral PNA-DNA duplex  

Microsoft Academic Search

Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton. They bind DNA and RNA with high specificity and selectivity, leading to PNA-RNA and PNA-DNA hybrids more stable than the corresponding nucleic acid complexes. The binding affinity and selectivity of PNAs for nucleic acids can be modified by the introduction of

Valeria Menchise; Giuseppina de Simone; Tullia Tedeschi; Roberto Corradini; Stefano Sforza; Rosangela Marchelli; Domenica Capasso; Michele Saviano; Carlo Pedone

2003-01-01

426

FIT probes: Peptide nucleic acid probes with a fluorescent base surrogate enable real-time DNA quantification and single nucleotide polymorphism discovery  

Microsoft Academic Search

The ability to accurately quantify specific nucleic acid molecules in complex biomolecule solutions in real time is important in diagnostic and basic research. Here we describe a DNA–PNA (peptide nucleic acid) hybridization assay that allows sensitive quantification of specific nucleic acids in solution and concomitant detection of select single base mutations in resulting DNA–PNA duplexes. The technique employs so-called FIT

Elke Socher; Dilip V. Jarikote; Andrea Knoll; Lars Röglin; Jens Burmeister; Oliver Seitz

2008-01-01

427

Structural basis of nucleic acid binding by Nicotiana tabacum glycine-rich RNA-binding protein: implications for its RNA chaperone function.  

PubMed

Glycine-rich RNA-binding proteins (GR-RBPs) are involved in cold shock response of plants as RNA chaperones facilitating mRNA transport, splicing and translation. GR-RBPs are bipartite proteins containing a RNA recognition motif (RRM) followed by a glycine-rich region. Here, we studied the structural basis of nucleic acid binding of full-length Nicotiana tabacum GR-RBP1. NMR studies of NtGR-RBP1 show that the glycine-rich domain, while intrinsically disordered, is responsible for mediating self-association by transient interactions with its RRM domain (NtRRM). Both NtGR-RBP1 and NtRRM bind specifically and with low micromolar affinity to RNA and single-stranded DNA. The solution structure of NtRRM shows that it is a canonical RRM domain. A HADDOCK model of the NtRRM-RNA complex, based on NMR chemical shift and NOE data, shows that nucleic acid binding results from a combination of stacking and electrostatic interactions with conserved RRM residues. Finally, DNA melting experiments demonstrate that NtGR-RBP1 is more efficient in melting CTG containing nucleic acids than isolated NtRRM. Together, our study supports the model that self-association of GR-RBPs by the glycine-rich region results in cooperative unfolding of non-native substrate structures, thereby enhancing its chaperone function. PMID:24957607

Khan, Fariha; Daniëls, Mark A; Folkers, Gert E; Boelens, Rolf; Saqlan Naqvi, S M; van Ingen, Hugo

2014-01-01

428

Shape matters: size-exclusion HPLC for the study of nucleic acid structural polymorphism  

PubMed Central

In recent years, an increasing number of reports have been focused on the structure and biological role of non-canonical nucleic acid secondary structures. Many of these studies involve the use of oligonucleotides that can often adopt a variety of structures depending on the experimental conditions, and hence change the outcome of an assay. The knowledge of the structure(s) formed by oligonucleotides is thus critical to correctly interpret the results, and gain insight into the biological role of these particular sequences. Herein we demonstrate that size-exclusion HPLC (SE-HPLC) is a simple yet surprisingly powerful tool to quickly and effortlessly assess the secondary structure(s) formed by oligonucleotides. For the first time, an extensive calibration and validation of the use of SE-HPLC to confidently detect the presence of different species displaying various structure and/or molecularity, involving >110 oligonucleotides forming a variety of secondary structures (antiparallel, parallel, A-tract bent and mismatched duplexes, triplexes, G-quadruplexes and i-motifs, RNA stem loops), is performed. Moreover, we introduce simple metrics that allow the use of SE-HPLC without the need for a tedious calibration work. We show that the remarkable versatility of the method allows to quickly establish the influence of a number of experimental parameters on nucleic acid structuration and to operate on a wide range of oligonucleotide concentrations. Case studies are provided to clearly illustrate the all-terrain capabilities of SE-HPLC for oligonucleotide secondary structure analysis. Finally, this manuscript features a number of important observations contributing to a better understanding of nucleic acid structural polymorphism. PMID:25143531

Largy, Eric; Mergny, Jean-Louis

2014-01-01

429

Detection of feline immunodeficiency virus (FIV) nucleic acids in FIV-seronegative cats.  

PubMed Central

A study was undertaken to determine the rate of viral transmission among naive specific-pathogen-free (SPF) cats living in close contact with feline immunodeficiency virus (FIV)-infected cats. Twenty SPF cats were housed in the same rooms with experimentally FIV-infected seropositive and virus culture-positive cats for 2 to 4 years and were monitored for the presence of FIV nucleic acids and antibodies. Only 1 of the 20 cats became seropositive and virus culture positive and developed signs of disease. Genomic DNA from bone marrow and peripheral blood mononuclear cells (PBMC) of 10 of 19 healthy-appearing seronegative cats became positive for FIV DNA by the polymerase chain reaction. Twenty-eight SPF cats housed as groups in separate quarters and never exposed to FIV-infected cats were uniformly negative for FIV DNA. FIV RNA transcripts were detected in concanavalin A-stimulated PBMC cultures from 4 of 10 FIV DNA-positive, seronegative cats by in situ hybridization. PBMC from three of four naive SPF cats acquired FIV nucleic acids after the cats were transfused with blood and bone marrow from FIV genome-positive, seronegative donors. Three of five FIV-seronegative cats housed for years with naturally FIV-infected cats in a private household were also found to harbor FIV DNA, indicating that the same phenomenon occurred in the field. These findings demonstrate that cats living in close contact with FIV-infected seropositive cats can acquire FIV nucleic acids without developing detectable levels of serum antibodies or disease. Images PMID:1318395

Dandekar, S; Beebe, A M; Barlough, J; Phillips, T; Elder, J; Torten, M; Pedersen, N

1992-01-01

430

Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase  

PubMed Central

Activation-induced deaminase (AID), a member of the larger AID/APOBEC family, is the key catalyst in initiating antibody somatic hypermutation and class-switch recombination. The DNA deamination model accounting for AID’s functional role posits that AID deaminates genomic deoxycytosine bases within the immunoglobulin locus, activating downstream repair pathways that result in antibody maturation. Although this model is well supported, the molecular basis for AID’s selectivity for DNA over RNA remains an open and pressing question, reflecting a broader need to elucidate how AID/APOBEC enzymes engage their substrates. To address these questions, we have synthesized a series of chimeric nucleic acid substrates and characterized their reactivity with AID. These chimeric substrates feature targeted variations at the 2?-position of nucleotide sugars, allowing us to interrogate the steric and conformational basis for nucleic acid selectivity. We demonstrate that modifications to the target nucleotide can significantly alter AID’s reactivity. Strikingly, within a substrate that is otherwise DNA, a single RNA-like 2?-hydroxyl substitution at the target cytosine is sufficient to compromise deamination. Alternatively, modifications that favor a DNA-like conformation (or sugar pucker) are compatible with deamination. AID’s closely related homolog APOBEC1 is similarly sensitive to RNA-like substitutions at the target cytosine. Inversely, with unreactive 2?-fluoro-RNA substrates, AID’s deaminase activity was rescued by introducing a trinucleotide DNA patch spanning the target cytosine and two nucleotides upstream. These data suggest a role for nucleotide sugar pucker in explaining the molecular basis for AID’s DNA selectivity and, more generally, suggest how other nucleic acid-modifying enzymes may distinguish DNA from RNA. PMID:23942124

Nabel, Christopher S.; Lee, Jae W.; Wang, Laura C.; Kohli, Rahul M.

2013-01-01

431

Boronic acid carrying (2-hydroxyethylmethacrylate)-based membranes for isolation of RNA  

Microsoft Academic Search

An affinity membrane that could be used for isolation of ribonucleic acid (RNA) from nucleic acid mixtures was synthesized by the radical copolymerization of 2-hydroxyethylmethacrylate (HEMA) with 4-vinylphenylboronic acid (VPBA). Potassium persulfate (KPS) and N,N,N?,N?-tetramethylethylenediamine (TEMED) were the redox initiator pair and N,N-methylenebisacrylamide (MBA) was the crosslinker in copolymerization conducted at room temperature (22°C). The effects of inclusion of VPBA

Serap Senel

2003-01-01

432

Design, Synthesis and Characterization of Nucleic Acid-Functionalized Gold Surfaces for Biomarker Detection  

PubMed Central

Nucleic acid-functionalized gold surfaces have been used extensively for the development of biological sensors. The development of an effective biomarker detection assay requires careful design, synthesis and characterization of probe components. In this feature article, we describe fundamental probe development constraints and provide a critical appraisal of the current methodologies and applications in the field. We discuss critical issues and obstacles that impede the sensitivity and reliability of the sensors to underscore the challenges that must be met to advance the field of biomarker detection. PMID:21905721

Adams, Nicholas M.; Jackson, Stephen R.; Haselton, Frederick R.; Wright, David W.

2014-01-01

433

METHODS FOR THE USE OF INDIUM AS AN ELECTRON STAIN FOR NUCLEIC ACIDS  

PubMed Central

Methods are presented for the staining of blocks of tissue with trivalent indium so that good contrast and good specificity for nucleic acids is achieved for the electron microscope. The tissue is fixed in organic fixative, dehydrated, subjected to reduction by lithium borohydride, acetylated by acetic anhydride, stained with trivalent indium dissolved in organic solvent, and embedded. The embedding material may be either Vestopal or butyl methacrylate especially handled to eliminate the "explosion" phenomenon. Numerous new problems encountered are discussed and a brief description of the findings is included. PMID:14005301

Watson, Michael L.; Aldridge, William G.

1961-01-01

434

The interpretation of Mg(2+) binding isotherms for nucleic acids using Poisson-Boltzmann theory.  

PubMed

Magnesium ions play a crucial role in the structural integrity and biological activity of nucleic acids. Experimental thermodynamic descriptions of Mg(2+) interactions with nucleic acids in solution have generally relied on the analyses of binding polynomials to estimate the energetic contributions of diffuse and site-bound ions. However, since ion binding is dominated by long-range electrostatic forces, such models provide only a phenomenological description of the experimental Mg(2+) binding data and provide little insight into the actual mechanism of the binding equilibria. Here, we present a rigorous theoretical framework based on the non-linear Poisson-Boltzmann (NLPB) equation for understanding diffuse ion interactions that can be used to interpret experimental Mg(2+) binding isotherms. As intuitively expected, in the NLPB model binding is simply the total accumulation of the ion around the nucleic acid. Comparing the experimental data to the calculated curves shows that the NLPB equation provides a remarkably accurate description of Mg(2+) binding to linear polynucleotides like DNA and poly(A x U) without any fitted parameters. In particular, the NLPB model explains two general features of magnesium binding; the strong dependence on univalent salt concentration, and its substantial anticooperativity. Each of these effects can be explained by changes in the Mg(2+) distribution around the polyion under different solution conditions. In order to more fully understand these different aspects of magnesium binding, the free energy of Mg(2+) binding, DeltaGMg, is calculated and partitioned into several salt-dependent contributions: the change in the electrostatic interaction free energy of the charges, DeltaDeltaGE.D (including Mg(2+)-phosphate, Mg(2+)-Mg(2+), Mg(2+)-Na(+), Na(+)-Na(+), Na(+)-phosphate interactions, and similar contributions for Cl(-)) and the cratic free energies of (re)organizing the MgCl2 and NaCl atmospheres, DeltaG(Mg)org and DeltaDeltaG(Na)org, respectively. For the systems studied here, DeltaGMg is strongly influenced by entropic free energy changes in the distributions of both NaCl and MgCl2, DeltaG(Mg)org and DeltaDeltaG(Na)org. From this analysis, we also raise the possibility that coions added with the magnesium salt might play an important role in the overall stability of nucleic acids under some conditions. PMID:10600372

Misra, V K; Draper, D E

1999-12-17

435

Targeting essential genes in Salmonella enterica serovar typhimurium with antisense peptide nucleic acid.  

PubMed

We investigated the capability of antisense peptide nucleic acids (PNAs) conjugated to the (KFF)(3)K cell-penetrating peptide to target possible essential genes (ligA, rpoA, rpoD, engA, tsf, and kdtA) in Salmonella enterica serovar Typhimurium and inhibit bacterial growth in vitro and in cell culture. All targeted PNA-based gene inhibition has shown great potency in gene expression inhibition in a sequence-specific and dose-dependent manner at micromolar concentrations. Among tested PNAs, the anti-rpoA and -rpoD PNAs showed the greatest potency. PMID:23006748

Soofi, Muhammad A; Seleem, Mohamed N

2012-12-01

436

Local electron correlation descriptions of the intermolecular stacking interactions between aromatic intercalators and nucleic acids  

NASA Astrophysics Data System (ADS)

Interaction energies for the binding of three intercalators to nucleic acid base pairs and base-pair steps are presented. Density fitting (DF) and local (L) correlation methods are employed, allowing use of basis sets appropriate for description of non-covalent interactions. In common with previous studies of stacking interactions, DF-LMP2 overestimates binding by as much as 50%. However, spin-component scaling (SCS) corrects for this effect, resulting in binding energies that support literature data obtained with small basis sets and/or density functional theory. The efficiency of this approach allows intercalators within base-pair steps to be studied, revealing substantial many body terms.

Grant Hill, J.; Platts, James A.

2009-09-01