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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

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

3

Use of methylated nucleic acid segments for isolating centromere DNA  

US Patent & Trademark Office Database

The invention provides efficient methods for the isolation of centromeres from potentially any organism. The methods may comprise the steps of: a) preparing a first sample of genomic DNA from a selected species; b) obtaining a plurality of methylated nucleic acid segments from the genomic DNA; and c) screening the methylated nucleic acid segments to identify a centromere nucleic acid sequence.

2003-11-18

4

Isolated menthone reductase and nucleic acid molecules encoding same  

DOEpatents

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

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

2013-04-23

5

Electricity-Free, Sequential Nucleic Acid and Protein Isolation  

PubMed Central

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4. By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

Pawlowski, David R.; Karalus, Richard J.

2012-01-01

6

Electricity-free, sequential nucleic acid and protein isolation.  

PubMed

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable (1). The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment (2). The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters (3). CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation(4). By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden. PMID:22635135

Pawlowski, David R; Karalus, Richard J

2012-01-01

7

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.

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

2013-01-01

8

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

9

Nucleic acid molecule  

US Patent & Trademark Office Database

The invention relates to an isolated nucleic acid molecule encoding a polypeptide capable of producing a triterpenoid hydrocarbon. The invention also relates to the encoded polypeptide, a vector comprising the nucleic acid molecule, a recombinant non-human organism comprising the nucleic acid molecule, and to methods of producing a triterpenoid hydrocarbon or an intermediate of biofuel using the nucleic acid molecule, polypeptide or recombinant organism.

2011-10-11

10

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

Microsoft Academic Search

A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid—based detection\\u000a of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic\\u000a acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling,\\u000a and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry

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

2010-01-01

11

Detection and isolation of nucleic acid sequences using a bifunctional hybridization probe  

DOEpatents

A method for detecting and isolating a target sequence in a sample of nucleic acids is provided using a bifunctional hybridization probe capable of hybridizing to the target sequence that includes a detectable marker and a first complexing agent capable of forming a binding pair with a second complexing agent. A kit is also provided for detecting a target sequence in a sample of nucleic acids using a bifunctional hybridization probe according to this method.

Lucas, Joe N. (San Ramon, CA); Straume, Tore (Tracy, CA); Bogen, Kenneth T. (Walnut Creek, CA)

2000-01-01

12

Comparison of nucleic acid-based detection of avian influenza H5N1 with virus isolation  

Microsoft Academic Search

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA\\/ECL) of avian influenza virus was compared with viral culture in embryonated chicken eggs. Virus was isolated from blood or anal swabs of chickens artificially infected with highly pathogenic avian influenza A\\/Chicken\\/Hong Kong\\/1000\\/97 (H5N1). Viral nucleic acid was detected in blood samples by NASBA\\/ECL immediately prior to death, whilst nucleic acid extracted from

Songhua Shan; Lung-Sang Ko; Richard A. Collins; Zhongliang Wu; Jiahua Chen; Ka-Yun Chan; Jun Xing; Lok-Ting Lau; Albert Cheung-Hoi Yub

2003-01-01

13

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

14

Automated nucleic acid isolation in viral molecular diagnostics: evaluation of the QIAsymphony SP.  

PubMed

The Qiagen QIAsymphony SP is a high-throughput (up to 96 samples per run), fully-automated nucleic acid isolation system. It was implemented in the authors' laboratory to cope with the high demand for pandemic H1N1 influenza testing in 2009. This study evaluated the QIAsymphony SP for viral nucleic acid isolation from quality control materials, pure cultures and various clinical specimens. The effect of varying sample volume on detection sensitivity was investigated using serial 10-fold dilutions of pure viral specimens and target nucleic acids were detected by real-time polymerase chain reaction (PCR) assays. Little variability in detection sensitivity was observed for all the viral targets tested, although variation in cycle threshold values was apparent in some cases. Importantly, pathogens were detectable over a broad concentration range and from diverse clinical specimens. Removal of PCR inhibitors was generally effective, as demonstrated by detection of viral nucleic acids and/or internal controls. The results demonstrate that the QIAsymphony SP is suitable for use in routine virology molecular diagnostics, and provides a high-throughput capacity, which is needed in peak seasons of infection or in centralised laboratories. PMID:22558800

Parham, N J; Parmar, S A; Kumar, N; Aliyu, S; Curran, M D; Zhang, H

2012-01-01

15

Surface Modification of Magnetic Silica Microspheres and Its Application to the Isolation of Plant Genomic Nucleic Acids  

Microsoft Academic Search

Solid-phase extraction of nucleic acids using magnetic particles has found widespread applications in biotechnological and biomedical research. On the basis of successful preparation of magnetic silica microspheres by a proprietary process, a further study was carried out on the surface modification of the magnetic silica microspheres and their applications in isolation of plant genomic nucleic acids. The surfaces of the

Zhi-Chao Zhang; Cui Yuan; Qian-Hong Wan

2007-01-01

16

A microchip for nucleic acid isolation and enrichment  

Microsoft Academic Search

This paper presents a microchip that effectively isolates and enriches target-binding single-stranded DNA (ssDNA) in a randomized DNA mixture using solid-phase extraction (SPE) and electrophoresis. The microchip consists of isolation and enrichment microchambers that are connected by a microchannel partially filled with agarose gel. Single-stranded DNA oligomers in the randomized mixture are captured via specific binding onto human immunoglobulin E

J. Kim; J. P. Hilton; K. A. Yang; R. Pei; K. Ennis; M. Stojanovic; Q. Lin

2012-01-01

17

Nucleic acid detection compositions  

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 structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

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

2008-08-05

18

Cleavage of nucleic acids  

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 structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

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

2000-01-01

19

Cleavage of nucleic acids  

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 structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

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

2010-11-09

20

Cleavage of nucleic acids  

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 structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

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

2007-12-11

21

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

22

Solid Phase Isolation of Proteins, Nucleic Acids and Other Macromolecules.  

National Technical Information Service (NTIS)

The invention is a method for the isolation of molecules of interest using tubes in which at least a portion of the inner walls of the tube are coated with microbeads that are coated with a capture reagent to bind the molecule of interest. The microbeads ...

F. von Lintig G. R. Boss J. C. Chen S. Hoffman S. B. Jones

2004-01-01

23

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.

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

2014-01-01

24

Method for preservation of nucleic acids  

US Patent & Trademark Office Database

A method for long-term maintenance of intact nucleic acid is provided. The method comprises introducing a nucleic acid containing receptacle into a cavity of a storage device. The nucleic acid containing receptacle comprises an amount of isolated vertebrate nucleic acid in a cavity of the receptacle, wherein either the receptacle or storage device is formed of a natural material. The nucleic acid is retained in the cavity by a fastener. A sealant is introduced into the aperture of the cavity of the device to form a nucleic acid storage device.

2001-07-10

25

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

26

Nucleic acid-based matrixes  

US Patent & Trademark Office Database

Various nucleic acid-based matrixes are provided, comprising nucleic acid monomers as building blocks, as well as nucleic acids encoding proteins, so as to produce novel biomaterials. Methods of utilizing such biomaterials include cell-free protein synthesis.

2013-07-16

27

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.

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

2013-01-01

28

Detection and isolation of nucleic acid sequences using competitive hybridization probes  

DOEpatents

A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided.

Lucas, Joe N. (San Ramon, CA); Straume, Tore (Tracy, CA); Bogen, Kenneth T. (Walnut Creek, CA)

1997-01-01

29

Detection and isolation of nucleic acid sequences using competitive hybridization probes  

DOEpatents

A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided. 7 figs.

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

1997-04-01

30

Nucleic Acid Cloning.  

National Technical Information Service (NTIS)

The present invention provides an improved system for linking nucleic acids to one another. In particular, the present invention provides techniques for producing DNA product molecules that may be easily and directly ligated to recipient molecules. The pr...

K. A. Jarrell V. W. Coljee W. Donahue S. Mikheeva

2001-01-01

31

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

32

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.

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

2011-01-01

33

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.

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

34

Composition for nucleic acid sequencing  

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

2008-08-26

35

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

36

Invasive cleavage of nucleic acids  

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 structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

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

2002-01-01

37

Invasive cleavage of nucleic acids  

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 structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

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

1999-01-01

38

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.

Chen, Xi

2010-01-01

39

Isolation of phosphatidylethanolamine as a solitary cofactor for prion formation in the absence of nucleic acids  

PubMed Central

Infectious prions containing the pathogenic conformer of the mammalian prion protein (PrPSc) can be produced de novo from a mixture of the normal conformer (PrPC) with RNA and lipid molecules. Recent reconstitution studies indicate that nucleic acids are not required for the propagation of mouse prions in vitro, suggesting the existence of an alternative prion propagation cofactor in brain tissue. However, the identity and functional properties of this unique cofactor are unknown. Here, we show by purification and reconstitution that the molecule responsible for the nuclease-resistant cofactor activity in brain is endogenous phosphatidylethanolamine (PE). Synthetic PE alone facilitates conversion of purified recombinant (rec)PrP substrate into infectious recPrPSc molecules. Other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol, were unable to facilitate recPrPSc formation in the absence of RNA. PE facilitated the propagation of PrPSc molecules derived from all four different animal species tested including mouse, suggesting that unlike RNA, PE is a promiscuous cofactor for PrPSc formation in vitro. Phospholipase treatment abolished the ability of brain homogenate to reconstitute the propagation of both mouse and hamster PrPSc molecules. Our results identify a single endogenous cofactor able to facilitate the formation of prions from multiple species in the absence of nucleic acids or other polyanions.

Deleault, Nathan R.; Piro, Justin R.; Walsh, Daniel J.; Wang, Fei; Ma, Jiyan; Geoghegan, James C.; Supattapone, Surachai

2012-01-01

40

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

41

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

42

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

43

A Simpler Nucleic Acid  

NASA Technical Reports Server (NTRS)

It has been supposed that for a nucleic acid analog to pair with RNA it must, like RNA, have a backbone with at least a sixatom repeat; a shorter backbone presumably would not stretch far enough to bind RNA properly. The Eschenmoser group has shown, however, that this first impression is incorrect.As they report in their new paper, Eschenmoser and co-workers ( I ) have now synthesized a substantial number of these polymers, which are called (L)-a-threofuranosyl oligonucleotides or TNAs. They are composed of bases linked to a threose sugar-phosphate backbone, with phosphodiester bonds connecting the nucleotides. The investigators discovered that pairs of complementary TNAs do indeed form stable Watson-Crick double helices and, perhaps more importantly, that TNAs form stable double helices with complementary RNAs and DNAs.

Orgel, Leslie

2000-01-01

44

Novel FDRG Protein and Nucleic Acid Molecules and Uses Therefor.  

National Technical Information Service (NTIS)

Novel FDRG polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated, full-length FDRG proteins, the invention further provides isolated FDRG fusion proteins, antigenic peptides and anti-FDRG antibodies. The invention also ...

B. M. Speigelman C. H. Yoon D. A. Holtzman

2004-01-01

45

Nucleic acid encoding human thyrotropin receptor  

US Patent & Trademark Office Database

Nucleic acids encoding human thyrotropin receptor and related methods are disclosed. The nucleic acids may have nucleotides deleted and/or replaced to modify the amino acid sequence of human thyrotropin receptor.

2004-06-08

46

Biopolymers: Protein and Nucleic Acids.  

National Technical Information Service (NTIS)

The work focuses on learning the principles that govern interactions between proteins and nucleic acids both DNA and RNA (specifically tRNA). With these principles as guides we are synthesizing peptides (of about 50 amino acids) that bind to specific regi...

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

1988-01-01

47

The Nucleic Acid Database (NDB)  

NSDL National Science Digital Library

Designed by John Westbrook, and housed at Rutgers University, the goal of the NDB is to assemble and distribute structural information about nucleic acids. The database contains the coordinates of nucleic acid-containing crystal structures, including a searchable atlas of structures, Protein Finder, a search-engine for locating protein structures in the PDB database, a macromolecular crystallographic information file, and archived reports about the structures contained in the database. This site provides information of general interest to researchers in the field, and develops and distributes standard geometry information for use in refinement and molecular modeling programs. Users can also subscribe to the NDB electronic newsletter.

1998-01-01

48

High speed nucleic acid sequencing  

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. Each type of labeled nucleotide comprises an acceptor fluorophore attached to a phosphate portion of the nucleotide such that the fluorophore is removed upon incorporation into a growing strand. Fluorescent signal is emitted via fluorescent resonance energy transfer between the donor fluorophore and the acceptor fluorophore as each nucleotide is incorporated into the growing strand. The sequence is deduced by identifying which base is being incorporated into the growing strand.

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

49

Replica amplification of nucleic acid arrays  

SciTech Connect

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

50

Control nucleic acids for multiple parameters  

US Patent & Trademark Office Database

Methods are provided for the amplification of at least a first and a second target nucleic acid that may be present in at least one fluid sample using an internal control nucleic acid for qualitative and/or quantitative purposes.

2013-12-17

51

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

52

Gold nanoparticles for nucleic Acid delivery.  

PubMed

Gold nanoparticles provide an attractive and applicable scaffold for delivery of nucleic acids. In this review, we focus on the use of covalent and noncovalent gold nanoparticle conjugates for applications in gene delivery and RNA-interference technologies. We also discuss challenges in nucleic acid delivery, including endosomal entrapment/escape and active delivery/presentation of nucleic acids in the cell. PMID:24599278

Ding, Ya; Jiang, Ziwen; Saha, Krishnendu; Kim, Chang Soo; Kim, Sung Tae; Landis, Ryan F; Rotello, Vincent M

2014-06-01

53

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

54

Nucleic acids encoding SOX10 promoter and methods to isolate SOX10 expressing cells  

US Patent & Trademark Office Database

DNA enhancer sequences are provided for use in constructs to identify early stage embryonic cells. The enhancer sequences can be used in parallel with short-hairpin RNA in a vector construct for endogenously regulated gene knockdowns. The disclosed enhancer sequences can be used to isolate a selected population of early stage embryonic cells.

2013-09-03

55

Efficient isolation of high quality nucleic acids from different tissues of Taxus baccata L  

Microsoft Academic Search

Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew\\u000a (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and ?-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation\\u000a were washed only with Chloroform: isoamyl

Abolghasem Abbasi Kejani; Sayed Ali Hosseini Tafreshi; Sayed Mojtaba Khayyam Nekouei; Mohammad Reza Mofid

2010-01-01

56

Nucleic Acid based logical systems.  

PubMed

Researchers increasingly visualize a significant role for artificial biochemical logical systems in biological engineering, much like digital logic circuits in electrical engineering. Those logical systems could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expression in vivo. Nucleic acids (NA), as carriers of genetic information with well-regulated and predictable structures, are promising materials for the design and engineering of biochemical circuits. A number of logical devices based on nucleic acids (NA) have been designed to handle various processes for technological or biotechnological purposes. This article focuses on the most recent and important developments in NA-based logical devices and their evolution from in vitro, through cellular, even towards in vivo biological applications. PMID:24692306

Han, Da; Kang, Huaizhi; Zhang, Tao; Wu, Cuichen; Zhou, Cuisong; You, Mingxu; Chen, Zhuo; Zhang, Xiaobing; Tan, Weihong

2014-05-12

57

Matrix isolation infrared studies of nucleic acid constituents. Part 4. Guanine and 9-methylguanine monomers and their keto—enol tautomerism  

NASA Astrophysics Data System (ADS)

Infrared absorption spectra have been studied for matrix isolated guanine and 9-methylguanine, which is a formal analogue of the natural nucleoside found in DNA and RNA. Three related compounds (isocytosine, 2-amino-5-chloropyrimidine and 2-dimethyl-amino-6-hydroxypurine) have also been examined. The spectra provide evidence for the existence of guanine and of 9-methylguanine as mixtures of the enol-amino and keto-amino tautomers, although the keto-amino tautomer is the only tautomeric form found in solution and in solid. The estimated enol—keto equilibrium constant fund in nitrogen matrices K = [E]/[K] is about 3.6 for guanine and 5.9 for 9-methylguanine. The significance of these results is evaluated in relation to the types of tautomers found in natural nucleic acids and to the concept of spontaneous and induced mutations caused by mis-pairing of the bases in the nucleic acids.

Szczepaniak, Krystyna; Szczesniak, Marian

1987-01-01

58

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

59

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

60

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

61

Peptide nucleic acids and their structural modifications.  

PubMed

Peptide (polyamide) analogues of nucleic acids (PNAs) make very promising groups of natural nucleic acid (NA) ligands and show many other interesting properties. Two types of these analogues may be highlighted as particularly interesting: the first, containing a polyamide with alternating peptide/pseudopeptide bonds as its backbone, consisting of N-(aminoalkyl)amino-acid units (type I), with nucleobases attached to the backbone nitrogen with the carboxyalkyl linker; and the second, containing a backbone consisting of amino-acid residues carrying the nucleobases in their side chains (type II). So far, these two groups have been studied most intensively. The paper describes main groups of peptide nucleic acids, as well as various other amino acid-derived nucleobase monomers or their oligomers, which were either studied in order to determine their hybridisation to nucleic acids, or only discussed with respect to their potential usefulness in the oligomerisation and nucleic acids binding. PMID:10698263

Falkiewicz, B

1999-01-01

62

Optimizing the specificity of nucleic acid hybridization  

Microsoft Academic Search

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

Sherry Xi Chen; David Yu Zhang; Peng Yin

2012-01-01

63

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.

Galindo-Murillo, Rodrigo; Bergonzo, Christina

2013-01-01

64

Finding and visualizing nucleic acid base stacking  

Microsoft Academic Search

Base stacking is one of the primary factors stabilizing nucleic acid structure. Yet, methods for locating stacking interactions in DNA and RNA are rare and methods for displaying stacking are rarer still. We present here simple, automated procedures to search nucleic acid molecules for base-base and base-oxygen stacking and to display these interactions graphically in a manner that readily conveys

H. A. Gabb; S. R. Sanghani; C. H. Robert; C. Prévost

1996-01-01

65

Inhibition and Facilitation of Nucleic Acid Amplification  

Microsoft Academic Search

Factors that inhibit the amplification of nucleic acids by PCR are present with target DNAs from many sources. The inhib- itors generally act at one or more of three essential points in the reaction in the following ways: they interfere with the cell lysis necessary for extraction of DNA, they interfere by nucleic acid degradation or capture, and they inhibit

IAN G. WILSON

1997-01-01

66

Rapid detection of fluoroquinolone resistance by isothermal chimeric primer-initiated amplification of nucleic acids from clinical isolates of Neisseria gonorrhoeae.  

PubMed

To ensure a complete response to fluoroquinolone therapy against Neisseria gonorrhoeae infections, rapid susceptibility determinations are required. We assessed a new approach, an isothermal chimeric primer-initiated amplification of nucleic acids (ICAN)/hybrid-chromatography method to detect rapidly fluoroquinolone resistance in N. gonorrhoeae. Comparison of the amplification results with fluoroquinolone minimum inhibitory concentrations (MICs), which were determined by an agar dilution method, showed that the new method accurately determined fluoroquinolone resistance in all ciprofloxacin- and/or gatifloxacin-resistant isolates, but agreed with results based on MICs in only 6 of 8 (75.0%) ciprofloxacin-susceptible and 7 of 12 (58.3%) gatifloxacin-susceptible isolates. Our results suggest that this method can rapidly and reliably detect point mutations in the gyrA gene as well as fluoroquinolone resistance in resistant isolates of N. gonorrhoeae. PMID:16278026

Horii, Toshinobu; Monji, Akio; Uemura, Keiichi; Nagura, Osanori

2006-06-01

67

Irreversible binding of acrylonitrile to nucleic acids.  

PubMed

1. [2,3-14C]Acrylonitrile was incubated with rat-liver microsomes, NADPH and either DNA, RNA or bovine serum albumin. Irreversible binding occurred to the macromolecular targets. Binding was lower when incubations were performed without microsomes. 2. Most of the 14C bound to DNA, RNA or polynucleotides (poly-A, poly-C, poly-G, poly-U) after incubation of [2,3-14C]acrylonitrile with rat-liver microsomes and 'conventional' re-isolation of the nucleic acids was removed from the macromolecular target when subsequently chromatographed on hydroxyapatite. 3. Radioactivity attached to DNA after prolonged non-enzymic incubations with [2,3-14C]acrylonitrile was also removed from the DNA by chromatography on hydroxyapatite. 4. When [2,3-14C]acrylonitrile was administered to rats (i.p.), incorporation of 14C into the natural bases of hepatic RNA was observed. In contrast with previous experiments with [1,2-14C]vinyl chloride, no radioactive [1-N6]etheno-adenine could be detected in RNA. 5. After administration of [2,3-14C]acrylonitrile to rats, hepatic DNA was isolated and hydrolysed by a modified enzymic procedure. Chromatography on PEI-cellulose showed two 14C peaks which did not co-chromatograph with any known standard. The amount of 14C in these presumed alkylation products was too low to allow chemical identification. 6. It is concluded that acrylonitrile, either itself or its metabolites, can alkylate nucleic acids. However, the extent of irreversible nucleic-acid binding is quantitatively much less than that observed with vinyl halides. PMID:6858196

Peter, H; Appel, K E; Berg, R; Bolt, H M

1983-01-01

68

Optimization of nucleic acid sequences.  

PubMed Central

Base sequence influences the structure, mechanics, dynamics, and interactions of nucleic acids. However, studying all possible sequences for a given fragment leads to a number of base combinations that increases exponentially with length. We present here a novel methodology based on a multi-copy approach enabling us to determine which base sequence favors a given structural change or interaction via a single energy minimization. This methodology, termed ADAPT, has been implemented starting from the JUMNA molecular mechanics program by adding special nucleotides, "lexides," containing all four bases, whose contribution to the energy of the system is weighted by continuously variable coefficients. We illustrate the application of this approach in the case of double-stranded DNA by determining the optimal sequences satisfying structural (B-Z transition), mechanical (intrinsic curvature), and interaction (ligand-binding) properties.

Lafontaine, I; Lavery, R

2000-01-01

69

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same  

DOEpatents

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

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

2012-10-16

70

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

71

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.

Lucas, Joe N. (San Ramon, CA) [San Ramon, CA; Straume, Tore (Tracy, CA) [Tracy, CA; Bogen, Kenneth T. (Walnut Creek, CA) [Walnut Creek, CA

1998-01-01

72

An ultrasensitive photoelectrochemical nucleic acid biosensor  

PubMed Central

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

Gao, Zhiqiang; Tansil, Natalia C.

2005-01-01

73

Sequence Comparison and Phylogeny of Nucleotide Sequence of Coat Protein and Nucleic Acid Binding Protein of a Distinct Isolate of Shallot virus X from India.  

PubMed

Shallot virus X (ShVX), a type species in the genus Allexivirus of the family Alfaflexiviridae has been associated with shallot plants in India and other shallot growing countries like Russia, Germany, Netherland, and New Zealand. Coat protein (CP) and nucleic acid binding protein (NB) region of the virus was obtained by reverse transcriptase polymerase chain reaction from scales leaves of shallot bulbs. The partial cDNA contained two open reading frames encoding proteins of molecular weights of 28.66 and 14.18 kDa belonging to Flexi_CP super-family and viral NB super-family, respectively. The percent identity and phylogenetic analysis of amino acid sequences of CP and NB region of the virus associated with shallot indicated that it was a distinct isolate of ShVX. PMID:23637504

Majumder, S; Baranwal, V K

2011-06-01

74

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

DOEpatents

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

Bavykin, Sergei (Darien, IL)

2011-01-18

75

Nucleic acid structures, energetics, and dynamics  

SciTech Connect

In 1953 DNA was shown to be a double helix of hydrogen-bonded complementary bases. Since then, knowledge of nucleic acid structures, thermodynamic stabilities, and dynamics of conformational changes has grown exponentially. This knowledge has led to the development of the biotechnology industry, the identification of plants and animals from a few cells, and many advances in the diagnosis and treatment of diseases. All the methods of physical chemistry have been used to characterize the primary structures (sequences), the secondary structures (base pairing), and the tertiary structures (folded 3D conformations) of the nucleic acids. The interactions of nucleic acids with themselves, with proteins, and with small-molecule ligands control their many functions. Novel methods are being developed to probe the structures and functions of nucleic acids. These include methods to study a single molecule and methods to select, amplify, and characterize one sequence among 10{sup 17} different sequences. 149 refs., 5 figs., 1 tab.

Tinoco, I. Jr. [Univ. of California, Berkeley, CA (United States)] [Univ. of California, Berkeley, CA (United States); [Lawrence Berkeley National Lab., CA (United States)

1996-08-01

76

Proteins and nucleic acids encoding same  

US Patent & Trademark Office Database

Disclosed are polypeptides and nucleic acids encoding same. Also disclosed are vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides, as well as methods for using same.

2007-10-02

77

Replica amplification of nucleic acid arrays  

DOEpatents

Disclosed are improved methods of making and using immobilized arrays of nucleic acids, particularly methods for producing replicas of such arrays. Included are methods for producing high density arrays of nucleic acids and replicas of such arrays, as well as methods for preserving the resolution of arrays through rounds of replication. Also included are methods which take advantage of the availability of replicas of arrays for increased sensitivity in detection of sequences on arrays. Improved methods of sequencing nucleic acids immobilized on arrays utilizing single copies of arrays and methods taking further advantage of the availability of replicas of arrays are disclosed. The improvements lead to higher fidelity and longer read lengths of sequences immobilized on arrays. Methods are also disclosed which improve the efficiency of multiplex PCR using arrays of immobilized nucleic acids.

Church, George M. (Brookline, MA); Mitra, Robi D. (Chestnut Hill, MA)

2010-08-31

78

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

79

Carbohydrate Polymers for Nonviral Nucleic Acid Delivery  

Microsoft Academic Search

\\u000a Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review,\\u000a we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the\\u000a recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran,

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

80

In vitro evolution of nucleic acids  

NASA Technical Reports Server (NTRS)

The author reviews recent published reports of in vitro selection and evolution of nucleic acids. These nucleic acids will bind to a target ligand or catalyze a specific chemical reaction. The terms aptamers and systematic evolution of ligands by exponential enrichment (SELEX) are explained. The review focuses on protein binders, small molecule binders, and ribozymes obtained by directed evolution. The reference list identifies articles of special or outstanding interest.

Joyce, G. F.; Miller, S. L. (Principal Investigator)

1994-01-01

81

Probe kit for identifying a base in a nucleic acid  

DOEpatents

Devices and techniques for hybridization of nucleic acids and for determining the sequence of nucleic acids. Arrays of nucleic acids are formed by techniques, preferably high resolution, light-directed techniques. Positions of hybridization of a target nucleic acid are determined by, e.g., epifluorescence microscopy. Devices and techniques are proposed to determine the sequence of a target nucleic acid more efficiently and more quickly through such synthesis and detection techniques.

Fodor, Stephen P. A. (Palo Alto, CA); Lipshutz, Robert J. (Palo Alto, CA); Huang, Xiaohua (Mt. View, CA)

2001-01-01

82

Method of Identifying a Base in a Nucleic Acid  

DOEpatents

Devices and techniques for hybridization of nucleic acids and for determining the sequence of nucleic acids. Arrays of nucleic acids are formed by techniques, preferably high resolution, light-directed techniques. Positions of hybridization of a target nucleic acid are determined by, e.g., epifluorescence microscopy. Devices and techniques are proposed to determine the sequence of a target nucleic acid more efficiently and more quickly through such synthesis and detection techniques.

Fodor, Stephen P. A. (Palo Alto, CA); Lipshutz, Robert J. (Palo Alto, CA); Huang, Xiaohua (Mt. View, CA)

1999-01-01

83

Hybridization and sequencing of nucleic acids using base pair mismatches  

DOEpatents

Devices and techniques for hybridization of nucleic acids and for determining the sequence of nucleic acids. Arrays of nucleic acids are formed by techniques, preferably high resolution, light-directed techniques. Positions of hybridization of a target nucleic acid are determined by, e.g., epifluorescence microscopy. Devices and techniques are proposed to determine the sequence of a target nucleic acid more efficiently and more quickly through such synthesis and detection techniques.

Fodor, Stephen P. A. (Palo Alto, CA); Lipshutz, Robert J. (Palo Alto, CA); Huang, Xiaohua (Mt. View, CA)

2001-01-01

84

Nucleic acids encoding plant defensins and methods of use thereof  

US Patent & Trademark Office Database

This invention relates to isolated nucleic acids encoding plant defensins. The invention also relates to the construction of a chimeric gene encoding all or a portion of the plant defensin, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of plant defensins in a transformed host cell.

2005-02-15

85

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

86

Soni-removal of nucleic acids from inclusion bodies.  

PubMed

Inclusion bodies (IBs) are commonly formed in Escherichia coli due to over expression of recombinant proteins in non-native state. Isolation, denaturation and refolding of these IBs is generally performed to obtain functional protein. However, during this process IBs tend to form non-specific interactions with sheared nucleic acids from the genome, thus getting carried over into downstream processes. This may hinder the refolding of IBs into their native state. To circumvent this, we demonstrate a methodology termed soni-removal which involves disruption of nucleic acid-inclusion body interaction using sonication; followed by solvent based separation. As opposed to conventional techniques that use enzymes and column-based separations, soni-removal is a cost effective alternative for complete elimination of buried and/or strongly bound short nucleic acid contaminants from IBs. PMID:24747565

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

2014-05-23

87

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

88

NUPACK: Analysis and design of nucleic acid systems.  

PubMed

The Nucleic Acid Package (NUPACK) is a growing software suite for the analysis and design of nucleic acid systems. The NUPACK web server (http://www.nupack.org) currently enables: Analysis: thermodynamic analysis of dilute solutions of interacting nucleic acid strands. Design: sequence design for complexes of nucleic acid strands intended to adopt a target secondary structure at equilibrium.Utilities: evaluation, display, and annotation of equilibrium properties of a complex of nucleic acid strands. NUPACK algorithms are formulated in terms of nucleic acid secondary structure. In most cases, pseudoknots are excluded from the structural ensemble. PMID:20645303

Zadeh, Joseph N; Steenberg, Conrad D; Bois, Justin S; Wolfe, Brian R; Pierce, Marshall B; Khan, Asif R; Dirks, Robert M; Pierce, Niles A

2011-01-15

89

Novel Biochip Platform for Nucleic Acid Analysis  

PubMed Central

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.

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

2012-01-01

90

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

91

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

92

Method for identifying and quantifying nucleic acid sequence aberrations  

DOEpatents

A method is disclosed for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe. 11 figs.

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

1998-07-21

93

Compositions and Methods Relating to Nucleic Acid Reference Standards.  

National Technical Information Service (NTIS)

The invention relates to novel nucleic acid reference standards comprising a nucleic acid comprising a known target sequence bound with a microparticulate binding agent where the binding agent includes liposomes, polyamines (e.g., nylon), siliceous compou...

C. A. Rundell J. Gordon

2004-01-01

94

Method for identifying and quantifying nucleic acid sequence aberrations  

DOEpatents

A method for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe.

Lucas, Joe N. (San Ramon, CA) [San Ramon, CA; Straume, Tore (Tracy, CA) [Tracy, CA; Bogen, Kenneth T. (Walnut Creek, CA) [Walnut Creek, CA

1998-01-01

95

Compositions and Methods for Systemic Nucleic Acid Sequence Delivery.  

National Technical Information Service (NTIS)

The present invention provides systemic nucleic acid sequence delivery without conventional systemic administration aids (SAAs). In certain embodiments, vascular permeability agents (VPAs), such as VEGF, are used in conjunction with nucleic acid viral vec...

J. M. Allen J. S. Chamberlain M. J. Blankinship P. Gregorevic

2004-01-01

96

Model studies of interactions between nucleic acids and proteins: hydrogen bonding of amides with nucleic acid bases.  

PubMed Central

The formation of hydrogen bonded complexes between nucleic acid bases and acetamide has been studied by nuclear magnetic resonance in CDC13 at different temperatures. Pairs of hydrogen bonds are formed when acetamide binds to nucleic acid bases. Thermodynamic parameters have been computed and compared to those obtained for the association of carboxylic acids with nucleic acid bases. The role of hydrogen bonded complexes in the association of proteins with nucleic acids is discussed.

Lancelot, G; Helene, C

1979-01-01

97

Encapsulated nanoparticles for nucleic acid delivery  

US Patent & Trademark Office Database

Methods and compositions for delivering agents (e.g., gene silencing agents) and molecules to cells using yeast cell wall particles are presented herein. Embodiments of the invention are particularly useful for the delivery of nucleic acids (e.g., siRNAs) to cells.

2013-03-05

98

Optical Melting Measurements of Nucleic Acid Thermodynamics  

PubMed Central

Optical melting experiments provide measurements of thermodynamic parameters for nucleic acids. These thermodynamic parameters are widely used in RNA structure prediction programs and DNA primer design software. This review briefly summarizes the theory and underlying assumptions of the method and provides practical details for instrument calibration, experimental design, and data interpretation.

Turner, Douglas H.

2014-01-01

99

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

100

Pyrophosphate-based method and apparatus for sequencing nucleic acids  

SciTech Connect

This patent describes a method for determining the nucleic acid sequence in a template nucleic acid polymer. It comprises: introducing the template nucleic acid polymer into a polymerization environment; successively providing to the polymerization environment a series of feedstocks; separately recovering each of the feedstocks from the polymerization environment; and measuring the amount of inorganic pyrophosphate.

Hyman, E.

1990-11-20

101

Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic Acid binding proteins.  

PubMed

Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation. PMID:24771851

Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

2014-06-01

102

In vitro selection of functional nucleic acids  

NASA Technical Reports Server (NTRS)

In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.

Wilson, D. S.; Szostak, J. W.

1999-01-01

103

Toward automated nucleic acid enzyme selection.  

PubMed

Methods for automation of nucleic acid selections are being developed. The selection of aptamers has been successfully automated using a Biomek 2000 workstation. Several binding species with nanomolar affinities were isolated from diverse populations. Automation of a deoxyribozyme ligase selection is in progress. The process requires eleven times more robotic manipulations than an aptamer selection. The random sequence pool contained a 5' iodine residue and the ligation substrate contained a 3' phosphorothioate. Initially, a manual deoxyribozyme ligase selection was performed. Thirteen rounds of selection yielded ligators with a 400-fold increase in activity over the initial pool. Several difficulties were encountered during the automation of DNA catalyst selection, including effectively washing bead-bound DNA, pipetting 50% glycerol solutions, purifying single strand DNA, and monitoring the progress of the selection as it is performed. Nonetheless, automated selection experiments for deoxyribozyme ligases were carried out starting from either a naive pool or round eight of the manually selected pool. In both instances, the first round of selection revealed an increase in ligase activity. However, this activity was lost in subsequent rounds. A possible cause could be mispriming during the unmonitored PCR reactions. Potential solutions include pool redesign, fewer PCR cycles, and integration of a fluorescence microtiter plate reader to allow robotic 'observation' of the selections as they progress. PMID:11688716

Sooter, L J; Riedel, T; Davidson, E A; Levy, M; Cox, J C; Ellington, A D

2001-09-01

104

Nucleic acid analysis using terminal-phosphate-labeled nucleotides  

SciTech Connect

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

105

Spectrofluorimetric study of the binding of codeine to nucleic acids  

NASA Astrophysics Data System (ADS)

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

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

2009-06-01

106

Detection of nucleic acids by multiple sequential invasive cleavages  

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

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

2012-10-16

107

Detection of nucleic acids by multiple sequential invasive cleavages  

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

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

1999-01-01

108

Detection of nucleic acids by multiple sequential invasive cleavages 02  

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

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

2002-01-01

109

Nonviral approach for targeted nucleic acid delivery.  

PubMed

Despite their relatively lower efficiency, nonviral approaches are emerging as safer alternatives in gene therapy to viral vectors. Delivery of nucleic acids to the target site is an important factor for effective gene expression (plasmid DNA) or knockdown (siRNA) with minimal side effects. Direct deposition at the target site by physical methods, including ultrasound, electroporation and gene gun, is one approach for local delivery. For less accessible sites, the development of carriers that can home into the target tissue is required. Cationic peptides, lipoplexes, polyplexes and nanoplexes have been used as carriers for delivery of nucleic acids. Targeting ligands, such as cell targeting peptides, have also been applied to decorate delivery vehicles in order to enhance their efficacy. This review focuses on delivery strategies and recent progress in non-viral carriers and their modifications to improve their performance in targeting and transfection. PMID:22320298

Jafari, M; Soltani, M; Naahidi, S; Karunaratne, D N; Chen, P

2012-01-01

110

Finding and visualizing nucleic acid base stacking.  

PubMed

Base stacking is one of the primary factors stabilizing nucleic acid structure. Yet, methods for locating stacking interactions in DNA and RNA are rare and methods for displaying stacking are rarer still. We present here simple, automated procedures to search nucleic acid molecules for base-base and base-oxygen stacking and to display these interactions graphically in a manner that readily conveys both the location and the quality of the interaction. The method makes no a priori assumptions about relative base positions when searching for stacking, nor does it rely on empirical energy functions. This is a distinct advantage for two reasons. First, the relative contributions of the forces stabilizing stacked bases are unknown. Second, the electrostatic and hydrophobic components of base stacking are both poorly defined by existing potential energy functions. PMID:8744567

Gabb, H A; Sanghani, S R; Robert, C H; Prévost, C

1996-02-01

111

Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes  

DOEpatents

A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

Ramsey, J. Michael (Knoxville, TN); Foote, Robert S. (Oak Ridge, TN)

2002-01-01

112

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.

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

2014-01-01

113

Transrenal nucleic acids: from proof of principle to clinical tests.  

PubMed

In spite of numerous publications on potential diagnostic application of circulating DNA and transrenal nucleic acid (Tr-NA) analysis, few, if any, tests based on this technology are available in clinical labs. This delay in test development and implementation is caused, at least in part, by the deficit in robust methods for isolation of short nucleic acid fragments from bodily fluids, as well as in techniques for analyzing these fragments. We have developed a new anion exchanger-based method for the isolation of cell-free nucleic acid fragments from large volumes of bodily fluids, and analyzed these fragments by PCR techniques specially designed to amplify "ultrashort" templates. The combination of these two techniques not only revealed the presence in urine of 10-150 bases or bp DNA and RNA fragments in addition to previously observed 150-200-bp DNA fragments and high molecular weight DNA, but also significantly increased the sensitivity of Tr-DNA detection. Additionally, we detected in urine a variety of miRNAs, including those excreted transrenally, thereby opening new diagnostic possibilities for Tr-NA analysis. PMID:18837928

Melkonyan, Hovsep S; Feaver, W John; Meyer, Erik; Scheinker, Vladimir; Shekhtman, Eugene M; Xin, Zhenghan; Umansky, Samuil R

2008-08-01

114

Circulating nucleic acids and apoptosis.  

PubMed

It is well documented that plasma contains DNA from tissues throughout the body, including developing fetuses, and tumors. A portion of this DNA crosses the kidney barrier and appears in urine (i.e., transrenal DNA). However, molecular, cellular, and physiological mechanisms of the circulating DNA phenomenon and renal clearance are in an early phase of investigation. Here, we discuss possible forms of circulating DNA, factors affecting representation of different tissues and genomic sequences in plasma DNA, possible mechanisms of renal DNA clearance, and technical problems encountered in DNA isolation from urine. We suggest that apoptotic cells are an important source of DNA in both plasma and urine. Further analysis of the data has led us to propose that a significant portion of circulating DNA can be represented in apoptotic bodies. PMID:11708486

Lichtenstein, A V; Melkonyan, H S; Tomei, L D; Umansky, S R

2001-09-01

115

Nucleic Acid Database: a Repository of Three-Dimensional Information about Nucleic Acids  

DOE Data Explorer

The Nucleic Acid Database (NDB) provides 3-D structural information about nucleic acids.  It is a relational database designed to facilitate the easy search for nucleic acid structures using any of the stored primary or derived structural features. Reports can then be created describing any properties of the selected structures and structures may be viewed in several different formats, including the mmCIF format, the NDB Atlas format, the NDB coordinate format, or the PDB coordinate format. Browsing structure images created directly from coordinates in the repository can also be done. More than 7000 structures have been released as of May 2014. This website also includes a number of specialized tools and interfaces. The NDB Project is funded by the National Institutes of Health and has been funded by the National Science Foundation and the Department of Energy in the past.

Berman, H.M.; Olson, W.K.; Beveridge, D.L.; Westbrook, J.; Gelbin, A.; Demeny, T.; Hsieh, S.H.; Srinivasan, A.R.; Schneider, B.

116

In vitro preparation of amelogenin nanoparticles carrying nucleic acids.  

PubMed

Amelogenin, a matrix protein involved in biomineralization of enamel, can self-assemble to form nanospheres in a pH-dependent manner. Nucleic acids (single-stranded, double-stranded, and plasmid DNA, as well as RNA) could be co-precipitated with amelogenin, demonstrating a strong binding of nucleic acids to amelogenin. The amounts of co-precipitated nucleic acids were analyzed and binding levels upto 90 ?g DNA/mg amelogenin was achieved. The co-precipitation could also be carried out in a bacterial cell homogenate, and no bacterial proteins were found in the amelogenin aggregates, suggesting specificity for nucleic acid binding. Dynamic light scattering showed that amelogenin nanosphere structure is maintained upon DNA binding with an upto 2.6 nm increase in diameter. The reported binding of nucleic acids to amelogenin can be explored practically for nucleic acid separation. PMID:24563322

Bonde, Johan; Bülow, Leif

2014-06-01

117

Diversity of the 47-kD HtrA Nucleic Acid and Translated Amino Acid Sequences from 17 Recent Human Isolates of Orientia  

PubMed Central

Abstract Orientia tsutsugamushi, the etiologic agent of potentially fatal scrub typhus, is characterized by a high antigenic diversity, which complicates the development of a broadly protective vaccine. Efficacy studies in murine and nonhuman primate models demonstrated the DNA vaccine candidate pKarp47, based upon the O. tsutsugamushi Karp 47-kD HtrA protein gene, to be a successful immunoprophylactic against scrub typhus. To characterize 47-kD HtrA protein diversity among human isolates of Orientia, we sequenced the full open reading frame (ORF) of the 47-kD HtrA gene and analyzed the translated amino acid sequences of 17 patient isolates from Thailand (n=13), Laos (n=2), Australia (n=1), and the United Arab Emirates (UAE) (n=1) and 9 reference strains: Karp (New Guinea), Kato (Japan), Ikeda (Japan), Gilliam (Burma), Boryong (Korea), TA763, TH1811 and TH1817 (Thailand), and MAK243 (China). The percentage identity (similarity) of translated amino acid sequences between 16 new isolates and 9 reference strains of O. tsutsugamushi ranged from 96.4% to 100% (97.4% to 100%). However, inclusion of the recently identified Orientia chuto sp. nov. reduced identity (similarity) values to 82.2% to 83.3% (90.4% to 91.4%). These results demonstrate the diversity of Orientia 47-kD HtrA among isolates encountered by humans and therefore provide support for the necessity of developing a broadly protective scrub typhus vaccine that takes this diversity into account.

Jiang, Ju; Paris, Daniel H.; Blacksell, Stuart D.; Aukkanit, Nuntipa; Newton, Paul N.; Phetsouvanh, Rattanaphone; Izzard, Leonard; Stenos, John; Graves, Stephen R.; Day, Nicholas P.J.

2013-01-01

118

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

119

Systems, compositions and methods for nucleic acid detection  

US Patent & Trademark Office Database

The invention relates to stretch measurements of nucleic acids and correlating those measurements to the extent of double- and single-stranded content of a nucleic acid of interest, and to compositions, systems, and devices related thereto. In preferred embodiments, one performs the stretch or elasticity measurements under conditions such that one can determine a nucleic acid sequence or the presence of an oligonucleotide in a sample.

2011-02-22

120

A Nucleic Acid Biosensor Based on Catalyzed Formation of Polyaniline  

Microsoft Academic Search

An ultrasensitive nucleic acid biosensor is described in this work. Following hybridization with a complementary nucleic acid and horseradish peroxidase (HRP) labeled oligonucleotide detection probe, a mixture of aniline\\/H2O2 in 0.10 M pH 4.0 phosphate buffer was applied to the biosensor. The hybridized polyanionic nucleic acid molecules serve as templates and HRP is the catalyst (aniline polymerization initiator). As a

Z. Gao; S. Rafea

2007-01-01

121

Nucleic acid delivery with chitosan hydroxybenzotriazole.  

PubMed

The objective of this study was to investigate the transfection efficiency of chitosan hydroxybenzotriazole (CS-HOBT) for in vitro nucleic acid delivery. The results revealed that CS-HOBT was able to condense with DNA/small interfering double-stranded RNA molecules (siRNA). Illustrated by agarose gel electrophoresis, complete complexes of CS-HOBT/DNA were formed at a weight ratio of above 3, whereas those of CS-HOBT/siRNA were formed at a weight ratio of above 4 (CS molecular weights [MWs] 20 and 45 kDa) and above 2 (CS MWs 200 and 460 kDa). Gel electrophoresis results indicated that binding of CS-HOBT and DNA or siRNA depended on the MW and weight ratio. The particle sizes of CS-HOBT/nucleic acid complexes were in nanosize range. The highest transfection efficiency of CS-HOBT/DNA complex was found at a weight ratio of 2, with the lowest CS MW of 20 kDa. The CS-HOBT-mediated siRNA silencing of the enhanced green fluorescent protein gene occurred maximally with 60% efficiency. The CS-HOBT/siRNA complex with the lowest CS MW of 20 kDa at a weight ratio of 80 showed the strongest inhibition of gene expression. For cytotoxicity studies, over 80% the average cell viabilities of the complexes were observed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. This study suggests that CS-HOBT is straightforward to prepare, is safe, and exhibits significantly improved nucleic acid delivery potential in vitro. PMID:20420543

Opanasopit, Praneet; Techaarpornkul, Sunee; Rojanarata, Theerasak; Ngawhirunpat, Tanasait; Ruktanonchai, Uracha

2010-06-01

122

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

123

The Nucleic Acid Database: new features and capabilities  

PubMed Central

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.

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

2014-01-01

124

Study on the interaction between nucleic acids and cationic surfactants.  

PubMed

The interactions of nucleic acids and cationic surfactants (cetylpyridine bromide (CPB) and cetyltrimethylammonium bromide (CTMAB)) in aqueous solution have been studied using the techniques of resonance light scattering (RLS) spectroscopy, the absorption spectroscopy, zeta potential assay and NMR assignment measurement. It is considered that CPB or CTMAB can assemble on the surface of nucleic acid via electrostatic and hydrophobic forces, which results in the formation of large associate of nucleic acid-cationic surfactant and RLS enhancement of nucleic acid. Besides these forces, the pi-pi stacking force between CPB and nucleic acid also exists in the associate. In comparison with CTMAB, CPB has larger enhancement on RLS of nucleic acid, which is attributed to that the enhancement of the former is only due to the absorption of the bases of nucleic acid, while the enhancement of the latter is own to the synergetic resonance caused by the absorption of both bases of nucleic acid and the pyridyl in CPB. These results have important implication for understanding the influence of surfactants on nucleic acid functionality in life science. PMID:15261091

Liu, Rutao; Yang, Jinghe; Sun, Changxia; Wu, Xia; Li, Lei; Su, Benyu

2004-03-01

125

Nucleic acid extraction from complex matrices  

US Patent & Trademark Office Database

The present disclosure describes an adsorbent and exemplary protocols for extracting nucleic acids, such as DNA and RNA, from complex matrices, such as stool samples and water samples. The adsorbent is activated charcoal coated with a material such as polyvinylpyrrolidone, dextran, or coconut flours. The adsorbent may be used in microcentrifuge spin columns, where it may be present as a slurry in a storage solution. The sample may be prepared by vortexing in a buffer solution, centrifuging, adding a protease to the supernatant, and passing the supernatant through a microcentrifuge spin column containing coated activated charcoal. The key components, including buffer, protease, and spin columns, may be packaged in a kit.

2013-11-05

126

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

127

Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino  

Microsoft Academic Search

Nucleic acid analogs and mimics are commonly the modifications of native nucleic acids at the nucleobase, the sugar ring, or the phosphodiester backbone. Many forms of promising nucleic acid analogs and mimics are available, such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos. LNAs, PNAs, and morpholinos can form both duplexes and triplexes and have improved biostability.

Shantanu Karkare; Deepak Bhatnagar

2006-01-01

128

EGVII endoglucanase and nucleic acids encoding the same  

DOEpatents

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

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

2008-11-11

129

EGVII endoglucanase and nucleic acids encoding the same  

DOEpatents

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

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

2012-02-14

130

EGVI endoglucanase and nucleic acids encoding the same  

DOEpatents

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

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

2008-04-01

131

EGVII endoglucanase and nucleic acids encoding the same  

DOEpatents

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

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

2009-05-05

132

EGVI endoglucanase and nucleic acids encoding the same  

DOEpatents

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

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

2010-10-05

133

EGVI endoglucanase and nucleic acids encoding the same  

DOEpatents

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

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

2010-10-12

134

EGVII endoglucanase and nucleic acids encoding the same  

DOEpatents

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

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

2013-07-16

135

Cell-free nucleic acids as biomarkers in cancer patients  

Microsoft Academic Search

DNA, mRNA and microRNA are released and circulate in the blood of cancer patients. Changes in the levels of circulating nucleic acids have been associated with tumour burden and malignant progression. In the past decade a wealth of information indicating the potential use of circulating nucleic acids for cancer screening, prognosis and monitoring of the efficacy of anticancer therapies has

Heidi Schwarzenbach; Dave S. B. Hoon; Klaus Pantel

2011-01-01

136

Chlorination effect on the fluorescence of nucleic acid staining dyes  

Microsoft Academic Search

An alternative to culture methods for the control of drinking water disinfection would use fluorescent dyes that could evidence the nucleic acid damages provoked by sodium hypochlorite treatment. The two dyes selected in this study, SYBR Green II RNA gel stain and TOTO-1 iodide, efficiently stain nucleic acids (DNA and RNA) and quite poorly the other biomolecules considered (Bovine serum

M. H. Phe; M. Dossot; J. C. Block

2004-01-01

137

Solid phase sequencing of double-stranded nucleic acids  

DOEpatents

This invention relates to methods for detecting and sequencing of target double-stranded nucleic acid sequences, to nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probe comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include nucleic acids in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated determination of molecular weights and identification of the target sequence.

Fu, Dong-Jing (Waltham, MA); Cantor, Charles R. (Boston, MA); Koster, Hubert (Concord, MA); Smith, Cassandra L. (Boston, MA)

2002-01-01

138

Nucleic acids encoding metal uptake transporters and their uses  

DOEpatents

The invention provides LCT1 nucleic acids which encode metal ion uptake transporters. The invention also provides methods of modulating heavy metal and alkali metal uptake in plants. The methods involve producing transgenic plants comprising a recombinant expression cassette containing an LCT1 nucleic acid linked to a plant promoter.

Schroeder, Julian I. (La Jolla, CA); Antosiewicz, Danuta M. (Warsaw, PL); Schachtman, Daniel P. (Tranmere, AU); Clemens, Stephan (San Diego, CA)

1999-01-01

139

Imperfectly matched nucleic acid complexes and their biochemical manifestation  

Microsoft Academic Search

The review is devoted to the analysis of experimental data on the selectivity of the interaction of nucleic acid with antisense oligonucleotides and their derivatives, which lead to the prospect of achieving a highly selective influence on many biochemical and molecular-genetic processes in living organisms. Theoretical estimates of the level of specificity of the interactions of nucleic acids and the

M. A. Zenkova; G. G. Karpova

1993-01-01

140

Nucleic Acid Amplification Testing in Suspected Child Sexual Abuse  

ERIC Educational Resources Information Center

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

Esernio-Jenssen, Debra; Barnes, Marilyn

2011-01-01

141

Purine and pyrimidine contents of some desoxypentose nucleic acids.  

PubMed

The distribution of purines and pyrimidines in desoxypentose nucleic acids prepared from a variety of animal and plant sources has been studied. 1. The nucleic acids were prepared from calf thymus, calf kidney, sheep spleen, horse spleen, chicken erythrocyte, turtle erythrocyte, trout sperm, shad testes, sea urchin sperm, wheat germ, and Pneumococcus Type III. 2. Separate hydrolyses were carried out for the determination of purines and pyrimidines. These procedures permitted nearly quantitative recovery of nucleic acid phosphorus in many of the preparations examined. 3. In the case of those preparations where a quantitative recovery was obtained it can be concluded that no bases other than adenine, guanine, thymine, and cytosine were present in appreciable amounts. 4. The distribution of purines and pyrimidines in all the nucleic acids studied renders the tetranucleotide hypothesis untenable. 5. The results of the analyses have indicated no great differences in the composition of these nucleic acids with respect to purines and pyrimidines. PMID:15422104

DALY, M M; ALLFREY, V G; MIRSKY, A E

1950-05-20

142

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

PubMed

Proper design of nucleic acid sequences is crucial for many applications. We have previously established a thermodynamics-based quantitative model to help design aptamer-based nucleic acid probes by predicting equilibrium concentrations of all interacting species. To facilitate customization of this thermodynamic model for different applications, here we present a generic and easy-to-use platform to implement the algorithm of the model with Microsoft(®) Excel formulas and VBA (Visual Basic for Applications) macros. Two Excel spreadsheets have been developed: one for the applications involving only nucleic acid species, the other for the applications involving both nucleic acid and non-nucleic acid species. The spreadsheets take the nucleic acid sequences and the initial concentrations of all species as input, guide the user to retrieve the necessary thermodynamic constants, and finally calculate equilibrium concentrations for all species in various bound and unbound conformations. The validity of both spreadsheets has been verified by comparing the modeling results with the experimental results on nucleic acid sequences reported in the literature. This Excel-based platform described here will allow biomedical researchers to rationalize the sequence design of nucleic acid probes using the thermodynamics-based modeling even without relevant theoretical and computational skills. PMID:23849929

Zou, Jiaqi; Li, Na

2013-09-01

143

Cyclopentane-Peptide Nucleic Acids for Qualitative, Quantitative, and Repetitive Detection of Nucleic Acids  

PubMed Central

We report the development of chemically-modified peptide nucleic acids (PNAs) as probes for qualitative and quantitative detection of DNA. The remarkable stability of PNAs toward enzymatic degradation makes this class of molecules ideal to develop as part of a diagnostic device that can be used outside of a laboratory setting. Using an enzyme-linked reporter assay, we demonstrate that excellent levels of detection and accuracy for anthrax DNA can be achieved using PNA probes with suitable chemical components designed into the probe. In addition, we report on DNA-templated crosslinking of PNA probes as a way to preserve genetic information for repetitive and subsequent analysis. This report is the first detailed examination of the qualitative and quantitative properties of chemically-modified PNA for nucleic acid detection and provides a platform for studying and optimizing PNA probes prior to incorporation into new technological platforms.

Micklitsch, Christopher M.; Oquare, Bereket Yemane; Zhao, Chao; Appella, Daniel H.

2012-01-01

144

Nucleic acid recognition by tandem helical repeats.  

PubMed

Protein domains constructed from tandem ?-helical repeats have until recently been primarily associated with protein scaffolds or RNA recognition. Recent crystal structures of human mitochondrial termination factor MTERF1 and Bacillus cereus alkylpurine DNA glycosylase AlkD bound to DNA revealed two new superhelical tandem repeat architectures capable of wrapping around the double helix in unique ways. Unlike DNA sequence recognition motifs that rely mainly on major groove read-out, MTERF and ALK motifs locate target sequences and aberrant nucleotides within DNA by resculpting the double-helix through extensive backbone contacts. Comparisons between MTERF and ALK repeats, together with recent advances in ssRNA recognition by Pumilio/FBF (PUF) domains, provide new insights into the fundamental principles of protein-nucleic acid recognition. PMID:22154606

Rubinson, Emily H; Eichman, Brandt F

2012-02-01

145

Nucleic Acid Recognition by Tandem Helical Repeats  

PubMed Central

Protein domains constructed from tandem ?-helical repeats have until recently been primarily associated with protein scaffolds or RNA recognition. Recent crystal structures of human mitochondrial termination factor MTERF1 and Bacillus cereus alkylpurine DNA glycosylase AlkD bound to DNA revealed two new superhelical tandem repeat architectures capable of wrapping around the double helix in unique ways. Unlike DNA sequence recognition motifs that rely mainly on major groove read-out, MTERF and ALK motifs locate target sequences and aberrant nucleotides within DNA by resculpting the double-helix through extensive backbone contacts. Comparisons between MTERF and ALK repeats, together with recent advances in ssRNA recognition by Pumilio/FBF (PUF) domains, provide new insights into the fundamental principles of protein-nucleic acid recognition.

Rubinson, Emily H.; Eichman, Brandt F.

2013-01-01

146

Nucleic acid sensor for insecticide detection.  

PubMed

Nucleic acid sensor based on polyaniline (PANI) has been fabricated by covalently immobilizing double stranded calf thymus (dsCT) DNA onto perchlorate (ClO(-) (4))-doped PANI film deposited onto indium-tin-oxide (ITO) glass plate using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) chemistry. These dsCT-DNA-PANI-ClO(4)/ITO and PANI-ClO(4)/ITO electrodes have been characterized using square wave voltammetry, electrochemical impedance, scanning electron microscopy (SEM) and Fourier-transform-infrared (FTIR) measurements. This disposable dsCT-DNA-PANI-ClO(4)/ITO bioelectrode, stable for about 4 months, can be used to detect cypermethrin (0.005 ppm) and trichlorfon (0.01 ppm) in 30 and 60 s, respectively. PMID:18446886

Solanki, Pratima R; Prabhakar, Nirmal; Pandey, M K; Malhotra, B D

2008-01-01

147

Nucleic Acid Database: a Repository of Three-Dimensional Information about Nucleic Acids  

DOE Data Explorer

The Nucleic Acid Database (NDB) provides 3-D structural information about nucleic acids.  It is a relational database designed to facilitate the easy search for nucleic acid structures using any of the stored primary or derived structural features. Reports can then be created describing any properties of the selected structures and structures may be viewed in several different formats, including the mmCIF format, the NDB Atlas format, the NDB coordinate format, or the PDB coordinate format. Browsing the images of the structures can also be done through the galleries of the X-ray Atlas or the NMR Atlas, available on this website. The images are created directly from coordinates in the NDB repository.  More than 3500 structures can be searched, viewed, and included in preformatted reports.This website also includes a number of specialized tools and interfaces. The NDB maintains the mmCIF Web site (macromolecular Crystallographic Information File), the IUCr-approved data representation for macromolecular structures. (Specialized Interface)

Berman, H.M.; Olson, W.K.; Beveridge, D.L.; Westbrook, J.; Gelbin, A.; Demeny, T.; Hsieh, S.H.; Srinivasan, A.R.; Schneider, B.

148

Nucleic acid-binding polymers as anti-inflammatory agents  

PubMed Central

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

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

2011-01-01

149

THE NUCLEIC ACID AND CARBOHYDRATE OF INFLUENZA VIRUS  

PubMed Central

Both ribonucleic and desoxyribonucleic acids have been obtained from purified particles of PR8 influenza virus. These particles were also found by extraction with formamide to contain carbohydrate in addition to that of the nucleic acids. Carbohydrate-rich fractions, essentially devoid of nucleic acid, were obtained not only from the particles representing PR8 virus but from those of Lee influenza virus as well. The carbohydrate in each case appeared to be a polysaccharide composed of mannose, galactose, and glucosamine units.

Knight, C. A.

1947-01-01

150

Oligonucleoside alkyl or arylphosphonate derivatives capable of crosslinking with or cleaving nucleic acids  

DOEpatents

A composition for inactivating a target nucleic acid which comprises an oligonucleoside alkyl or arylphosphonate analogue which is complementary to the sequence of the target nucleic acid and includes a functional group which reacts with the target nucleic acid to render the target nucleic acid inactive or nonfunctional.

Miller, Paul S. (Baltimore, MD); Ts'o, Paul O.P. (Lutherville, MD)

1999-06-15

151

Oligonucleoside alkyl or arylphosphonate derivatives capable of crosslinking with or cleaving nucleic acids  

DOEpatents

A composition for inactivating a target nucleic acid which comprises an oligonucleoside alkyl or arylphosphonate analogue which is complementary to the sequence of the target nucleic acid is provided. It includes a functional group which reacts with the target nucleic acid to render the target nucleic acid inactive or nonfunctional. 16 figs.

Miller, P.S.; Ts'o, P.O.P.

1999-06-15

152

Nucleic Acid Cloning (PAT-APPL-11-132 356).  

National Technical Information Service (NTIS)

The present invention provides an improved system for linking nucleic acids to one another. In particular, the present invention provides techniques for producing DNA product molecules that may be easily and directly ligated to recipient molecules. The pr...

K. A. Jarrell S. Mikheeva V. W. Coljee W. Donahue

2005-01-01

153

Nucleic acid duplexes incorporating a dissociable covalent base pair  

NASA Technical Reports Server (NTRS)

We have used molecular modeling techniques to design a dissociable covalently bonded base pair that can replace a Watson-Crick base pair in a nucleic acid with minimal distortion of the structure of the double helix. We introduced this base pair into a potential precursor of a nucleic acid double helix by chemical synthesis and have demonstrated efficient nonenzymatic template-directed ligation of the free hydroxyl groups of the base pair with appropriate short oligonucleotides. The nonenzymatic ligation reactions, which are characteristic of base paired nucleic acid structures, are abolished when the covalent base pair is reduced and becomes noncoplanar. This suggests that the covalent base pair linking the two strands in the duplex is compatible with a minimally distorted nucleic acid double-helical structure.

Gao, K.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

1999-01-01

154

Immunochemistry, Structure and Function of Nucleic Acids: Abstracts.  

National Technical Information Service (NTIS)

Thirty-five abstracts on the immunochemistry, structure and function of nucleic acids are presented. The abstracts discuss the immunochemistry of deoxyribonucleotides, DNA and RNA; gene activity and left-handed helical DNA; modified nucleotides in transfe...

1987-01-01

155

Engineered nucleic acids encoding a modified erythropoietin and their expression  

US Patent & Trademark Office Database

Provided are formulations, compositions and methods for delivering biological moieties such as modified nucleic acids into cells to modulate protein expression. Such compositions and methods include the delivery of biological moieties, and are useful for production of proteins.

2014-04-29

156

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.

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

2013-01-01

157

Sharing and archiving nucleic acid structure mapping data  

PubMed Central

Nucleic acids are particularly amenable to structural characterization using chemical and enzymatic probes. Each individual structure mapping experiment reveals specific information about the structure and/or dynamics of the nucleic acid. Currently, there is no simple approach for making these data publically available in a standardized format. We therefore developed a standard for reporting the results of single nucleotide resolution nucleic acid structure mapping experiments, or SNRNASMs. We propose a schema for sharing nucleic acid chemical probing data that uses generic public servers for storing, retrieving, and searching the data. We have also developed a consistent nomenclature (ontology) within the Ontology of Biomedical Investigations (OBI), which provides unique identifiers (termed persistent URLs, or PURLs) for classifying the data. Links to standardized data sets shared using our proposed format along with a tutorial and links to templates can be found at http://snrnasm.bio.unc.edu.

Rocca-Serra, Philippe; Bellaousov, Stanislav; Birmingham, Amanda; Chen, Chunxia; Cordero, Pablo; Das, Rhiju; Davis-Neulander, Lauren; Duncan, Caia D.S.; Halvorsen, Matthew; Knight, Rob; Leontis, Neocles B.; Mathews, David H.; Ritz, Justin; Stombaugh, Jesse; Weeks, Kevin M.; Zirbel, Craig L.; Laederach, Alain

2011-01-01

158

Effect of Berenil on Nucleic Acid Synthesis in Trypanosoma brucei.  

National Technical Information Service (NTIS)

The present study was undertaken to examine (a) the effect of diaminazene on the growth and cellular morphology of monomorphic bloodstream trypanosomes; (b) the effect of diaminazene on nucleic acid and protein synthesis of these trypanosomes; and (c) the...

M. S. Zahalsky A. C. Zahalsky

1979-01-01

159

Detection of Cytomegalovirus by Nucleic Acid Hybridization Techniques.  

National Technical Information Service (NTIS)

Nucleic acid hybridization techniques, RNA-DNA membrane hybridization, DNA-DNA renaturation kinetics analysis (Cot analysis) and in situ 3H cRNA-DNA cytohybridization, were developed and successfully applied to detection of human cytomegalovirus (CMV) gen...

J. S. Pagano E. S. Huang

1975-01-01

160

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.

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

2007-01-01

161

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.

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

2012-01-01

162

Changes of nucleic acids of wheat seedlings under spaceflight conditions  

NASA Technical Reports Server (NTRS)

The effects of space flight on the growth of wheat seedlings and their nucleic acid content were studied. It was shown that both space and ground seedlings have almost the same appearance, dry weight and nucleic acid content in the root, coleoptile and leaves. The only difference found is in the RNA and DNA content, which is twice as much in the ground seedling apices as in the space-grown seedlings.

Sytnyk, K. M.; Musatenko, L. I.

1983-01-01

163

Metal complex derivatives of peptide nucleic acids (PNA).  

PubMed

Peptide nucleic acid (PNA) is a non-cyclic pseudopeptide-nucleic acid structural mimic with promising applications within diagnostics and drug discovery. This review focuses on metal complex derivatives of PNA. Metal ions and their complexes display unique physical and chemical properties and offer the opportunity to introduce new labels and probes for bioanalytical and diagnostic applications of PNA, but also to modulate or to introduce new (for example catalytic) functions and biological activities. PMID:22210345

Krämer, Roland; Mokhir, Andriy

2012-01-01

164

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

165

[Ten years of nucleic acid testing: lessons and prospects].  

PubMed

Nucleic acid testing has been routinely performed in all blood donations in France since July 1st 2001. This is the story of a controversial decision. "The unacceptable HIV risk" in the context of the early 2000s influenced the decision. The results achieved over these past 10 years are analyzed given the expected progress of this new screening tool for infectious agents in transfusion. They confirm the relevance of models used by experts in 2000. Out of 22.3 million donations over the period (2001-2009), 22 donations have been rejected because of nucleic acid testing positive for hepatitis C virus (n=11) and human immunodeficiency virus (n=11). Nucleic acid testing has contributed to improve the functioning of the transfusion chain activities in order to ensure the availability of blood products. In terms of reactivity against emerging infectious agents, its role in the West Nile virus (WNV) outbreak is exemplary, but it did not play a similar role in crises of the same order. ALT determination has been stopped thanks to nucleic acid testing. The risk of contamination of the method by amplification products has been confirmed and caution is still required. Nucleic acid testing is being maintained and reached a new milestone in 2010 with the implementation of a full automated system, meanwhile pool screening was given up and hepatitis B virus screening became widespread. Nucleic acid tests will probably be revised when all blood products are pathogen-inactivated. PMID:21397544

Morel, P

2011-04-01

166

Towards Accurate Prediction of Protonation Equilibrium of Nucleic Acids  

PubMed Central

The role of protonated nucleotides in modulating the pH-dependent properties of nucleic acids is one of the emerging frontiers in the field of nucleic acid biology. The recent development of a constant pH molecular dynamics simulation (CPHMDMS?D) framework for simulating nucleic acids has provided a tool for realistic simulations of pH-dependent dynamics. We enhanced the CPHMDMS?D framework with pH-based replica exchange (pH-REX), which significantly improves the sampling of both titration and spatial coordinates. The results from our pKa calculations for the GAAA tetraloop, which was predicted with lower accuracy previously due to sampling challenges, demonstrates that pH-REX reduces the average unsigned error (AUE) to 0.7 pKa units, and the error of the most poorly predicted residue A17 was drastically reduced from 2.9 to 1.2 pKa unit. Lastly, we show that pH-REX CPHMDMS?D simulations can be used to identify the dominant conformation of nucleic acid structures in alternate pH environments. This work suggests that pH-REX CPHMDMS?D simulations provide a practical tool for predicting nucleic acid protonation equilibrium from first-principles, and offering structural and mechanistic insight into the study of pH-dependent properties of nucleic acids.

Goh, Garrett B.; Knight, Jennifer L.

2013-01-01

167

Characterization of metal ion-nucleic acid interactions in solution.  

PubMed

Metal ions are inextricably involved with nucleic acids due to their polyanionic nature. In order to understand the structure and function of RNAs and DNAs, one needs to have detailed pictures on the structural, thermodynamic, and kinetic properties of metal ion interactions with these biomacromolecules. In this review we first compile the physicochemical properties of metal ions found and used in combination with nucleic acids in solution. The main part then describes the various methods developed over the past decades to investigate metal ion binding by nucleic acids in solution. This includes for example hydrolytic and radical cleavage experiments, mutational approaches, as well as kinetic isotope effects. In addition, spectroscopic techniques like EPR, lanthanide(III) luminescence, IR and Raman as well as various NMR methods are summarized. Aside from gaining knowledge about the thermodynamic properties on the metal ion-nucleic acid interactions, especially NMR can be used to extract information on the kinetics of ligand exchange rates of the metal ions applied. The final section deals with the influence of anions, buffers, and the solvent permittivity on the binding equilibria between metal ions and nucleic acids. Little is known on some of these aspects, but it is clear that these three factors have a large influence on the interaction between metal ions and nucleic acids. PMID:22210334

Pechlaner, Maria; Sigel, Roland K O

2012-01-01

168

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

169

Formulated nucleic acid compositions and methods of administering the same for gene therapy  

US Patent & Trademark Office Database

Compositions and methods for administering nucleic acid compositions in vitro to cells in culture or in vivo to an organism whereby the uptake of nucleic acids is enhanced are provided. Various compositions, including those incorporating protective, interactive, non-condensing compounds, are utilized to protect and administered nucleic acid formulation, thereby prolonging the localized bioavailability of the administered nucleic acid and enhancing expression from the nucleic acid.

2003-02-04

170

Nucleic acid sequences and expression system relating to Enterococcus faecium for diagnostics and therapeutics  

US Patent & Trademark Office Database

The invention provides isolated polypeptide and nucleic acid sequences derived Enterococcus faecium that are useful in diagnosis and therapy of pathological conditions; antibodies against the polypeptides; and methods for the production of the polypeptides. The invention also provides methods for the detection, prevention and treatment of pathological conditions resulting from bacterial infection.

2003-06-24

171

Nucleic acid and polypeptide aptamers: a powerful approach to ligand discovery  

Microsoft Academic Search

Nucleic acid ligands against important targets in infectious, malignant and vascular diseases are continually being discovered by the process of in vitro evolution. Promising methods are being developed for the exploitation of these aptamers in biosensors and other diagnostic devices. Ribosome and mRNA display methods for isolating polypeptide aptamers from highly complex libraries have been developed and have the potential

William James

2001-01-01

172

Nucleotide Composition of Nucleic Acids of Fungi II. Deoxyribonucleic Acids  

PubMed Central

Storck, Roger (The University of Texas, Austin). Nucleotide composition of nucleic acids from fungi. II. Deoxyribonucleic acids. J. Bacteriol. 91:227–230. 1966.—The nucleotide composition of the deoxyribonucleic acids (DNA) present in extracts of 30 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 38 to 63% in a distribution composed of 9 species of zygomycetes, 14 of ascomycetes, and 9 each of deuteromycetes and basidiomycetes. The GC content ranges were: 38 to 48% for the zygomycetes, 38 to 54% for the ascomycetes, 47 to 62% for the deuteromycetes, and 44 to 63% for the basidiomycetes. The GC content ranged from 38 to 40% for four Mucor species. The base composition of fungal DNA appears, therefore, to have a taxonomic and phylogenetic significance.

Storck, Roger

1966-01-01

173

[Method for automated extraction and purification of nucleic acids and its implementation in microfluidic system].  

PubMed

A method and a microfluidic device for automated extraction and purification of nucleic acids from biological samples have been developed. The method involves disruption of bacterial cells and/or viral particles by combining enzymatic and chemical lysis procedures followed by solid-phase sorbent extraction and purification of nucleic acids. The procedure is carried out in an automated mode in a microfluidic module isolated from the outside environment, which minimizes contact of the researcher with potentially infectious samples and, consequently, decreases the risk of laboratory-acquired infections. The module includes reservoirs with lyophilized components for lysis and washing buffers; a microcolumn with a solid-phase sorbent; reservoirs containing water, ethanol, and water-ethanol buffer solutions for dissolving freeze-dried buffer components, rinsing the microcolumn, and eluting of nucleic acids; and microchannels and valves needed for directing fluids inside the module. The microfluidic module is placed into the control unit that delivers pressure, heats, mixes reagents, and flows solutions within the microfluidic module. The microfluidic system performs extraction and purification of nucleic acids with high efficiency in 40 min, and nucleic acids extracted can be directly used in PCR reaction and microarray assays. PMID:22808749

Mamaev, D D; Khodakov, D A; Dement'eva, E I; Filatov, I V; Iurasov, D A; Cherepanov, A I; Vasiliskov, V A; Smoldovskaia, O V; Zimenkov, D V; Griadunov, D A; Mikha?lovich, V M; Zasedatelev, A S

2011-01-01

174

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

175

Isolation and Detection of Enterovirus RNA from Large-Volume Water Samples by Using the NucliSens miniMAG System and Real-Time Nucleic Acid Sequence-Based Amplification  

PubMed Central

Concentration of water samples is a prerequisite for the detection of the low virus levels that are present in water and may present a public health hazard. The aim of this study was to develop a rapid, standardized molecular method for the detection of enteroviruses in large-volume surface water samples, using a concentration method suitable for the detection of infectious viruses as well as virus RNA. Concentration of water was achieved by a conventional filter adsorption-elution method and ultrafiltration, resulting in a 10,000-fold concentration of the sample. Isolation of virus RNA by a silica-based RNA extraction method was compared with the nonmagnetic and magnetic NucliSens RNA isolation methods. By using the silica-based RNA extraction method in two out of five samples, enterovirus RNA was detected, whereas four out of five samples were positive following RNA isolation with magnetic silica beads. Moreover, estimated RNA levels increased at least 100 to 500 times. Furthermore, we compared enterovirus detection by an in-house reverse transcription (RT)-PCR with a novel commercially available real-time nucleic acid sequence-based amplification (NASBA) assay. We found that the rapid real-time NASBA assay was slightly less sensitive than our in-house RT-PCR. The advantages, however, of a commercial real-time NASBA assay, like the presence of an internal control RNA, standardization, and enormous decrease in turnaround time, makes it an attractive alternative to RT-PCR.

Rutjes, Saskia A.; Italiaander, Ronald; van den Berg, Harold H. J. L.; Lodder, Willemijn J.; de Roda Husman, Ana Maria

2005-01-01

176

Isolation and detection of enterovirus RNA from large-volume water samples by using the NucliSens miniMAG system and real-time nucleic acid sequence-based amplification.  

PubMed

Concentration of water samples is a prerequisite for the detection of the low virus levels that are present in water and may present a public health hazard. The aim of this study was to develop a rapid, standardized molecular method for the detection of enteroviruses in large-volume surface water samples, using a concentration method suitable for the detection of infectious viruses as well as virus RNA. Concentration of water was achieved by a conventional filter adsorption-elution method and ultrafiltration, resulting in a 10,000-fold concentration of the sample. Isolation of virus RNA by a silica-based RNA extraction method was compared with the nonmagnetic and magnetic NucliSens RNA isolation methods. By using the silica-based RNA extraction method in two out of five samples, enterovirus RNA was detected, whereas four out of five samples were positive following RNA isolation with magnetic silica beads. Moreover, estimated RNA levels increased at least 100 to 500 times. Furthermore, we compared enterovirus detection by an in-house reverse transcription (RT)-PCR with a novel commercially available real-time nucleic acid sequence-based amplification (NASBA) assay. We found that the rapid real-time NASBA assay was slightly less sensitive than our in-house RT-PCR. The advantages, however, of a commercial real-time NASBA assay, like the presence of an internal control RNA, standardization, and enormous decrease in turnaround time, makes it an attractive alternative to RT-PCR. PMID:16000783

Rutjes, Saskia A; Italiaander, Ronald; van den Berg, Harold H J L; Lodder, Willemijn J; de Roda Husman, Ana Maria

2005-07-01

177

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.

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

2013-01-01

178

Methods And Devices For Characterizing Duplex Nucleic Acid Molecules  

DOEpatents

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

Akeson, Mark (Santa Cruz, CA); Vercoutere, Wenonah (Santa Cruz, CA); Haussler, David (Santa Cruz, CA); Winters-Hilt, Stephen (Santa Cruz, CA)

2005-08-30

179

Structural Requirements for the Procoagulant Activity of Nucleic Acids  

PubMed Central

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

Gansler, Julia; Jaax, Miriam; Leiting, Silke; Appel, Bettina; Greinacher, Andreas; Fischer, Silvia; Preissner, Klaus T.

2012-01-01

180

Nucleic acids electrotransfer in vivo: mechanisms and practical aspects.  

PubMed

Nucleic acids transfer has been steadily improving over the years and is slowly starting to fulfill its long awaited promises. In the beginning, viral approaches raised strong safety concerns that are now answered by various non-viral techniques. Among the physical approaches developed, nucleic acids electrotransfer is probably the one with the highest momentum. Here we review the present knowledge on the mechanistic and practical aspects of in vivo nucleic acids electrotransfer. For each step of this procedure we present different strategies that are used, with their advantages and drawbacks. As we report here, practical solutions have been found to overcome each limiting step in the procedure and to improve its outcome. Some crucial issues are beyond the application of the electric pulses itself, like the administration (i.e., in almost all of the cases, the injection) of the nucleic acids to the tissue or the body. High efficiency and safety are at reach if all the present knowledge and strategies are put to use. Electrotransfer is now a mature technique as proven by the fact that clinical trials using nucleic acids electrotransfer have already started within the past few years. PMID:20557285

Andre, Franck M; Mir, Lluis M

2010-08-01

181

Nucleic acid-based nanoengineering: novel structures for biomedical applications  

PubMed Central

Nanoengineering exploits the interactions of materials at the nanometre scale to create functional nanostructures. It relies on the precise organization of nanomaterials to achieve unique functionality. There are no interactions more elegant than those governing nucleic acids via Watson–Crick base-pairing rules. The infinite combinations of DNA/RNA base pairs and their remarkable molecular recognition capability can give rise to interesting nanostructures that are only limited by our imagination. Over the past years, creative assembly of nucleic acids has fashioned a plethora of two-dimensional and three-dimensional nanostructures with precisely controlled size, shape and spatial functionalization. These nanostructures have been precisely patterned with molecules, proteins and gold nanoparticles for the observation of chemical reactions at the single molecule level, activation of enzymatic cascade and novel modality of photonic detection, respectively. Recently, they have also been engineered to encapsulate and release bioactive agents in a stimulus-responsive manner for therapeutic applications. The future of nucleic acid-based nanoengineering is bright and exciting. In this review, we will discuss the strategies to control the assembly of nucleic acids and highlight the recent efforts to build functional nucleic acid nanodevices for nanomedicine.

Li, Hanying; LaBean, Thomas H.; Leong, Kam W.

2011-01-01

182

Conformational transitions in human translin enable nucleic acid binding.  

PubMed

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

Pérez-Cano, Laura; Eliahoo, Elad; Lasker, Keren; Wolfson, Haim J; Glaser, Fabian; Manor, Haim; Bernadó, Pau; Fernández-Recio, Juan

2013-11-01

183

Study of the interaction of nucleic acids with acridine orange-CTMAB and determination of nucleic acids at nanogram levels based on the enhancement of resonance light scattering  

NASA Astrophysics Data System (ADS)

The resonance light scattering (RLS) of acridine orange (AO) are greatly enhanced by both nucleic acid and cetyltrimethyl ammonium bromide (CTMAB). Based on this, nucleic acids can be sensitively determined. The interaction of AO with nucleic acid in the presence of CTMAB is studied by using RLS, absorption spectroscopy, zeta potential assay, transmission electron microscope (TEM) image and molecular molding. On its surface, nucleic acid acts as the template to promote the assembly of both AO and CTMAB in close proximity, which formatted large AO-nucleic acid-CTMAB association. Therefore, RLS intensity of cationic dye is greatly enhanced.

Liu, Rutao; Yang, Jinghe; Sun, Changxia; Li, Lei; Wu, Xia; Li, Zhengmin; Qi, Chuansong

2003-07-01

184

Modular Design and Construction of Nucleic Acid Molecules, Aptamer-Derived Nucleic Acid Constructs, RNA Scaffolds, their Expression, and Methods of Use.  

National Technical Information Service (NTIS)

The present invention relates to a nucleic acid molecule comprised of first and second nucleic acid elements that each bind a target molecule, and a three-way junction operably linking the first and second nucleic elements. Also disclosed is an RNA scaffo...

H. Shi J. T. Lis

2005-01-01

185

Synthesis and characterization of peptide nucleic acid platinum nanoclusters  

Microsoft Academic Search

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

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

2006-01-01

186

Nature and Magnitude of Aromatic Stacking of Nucleic Acid Bases  

SciTech Connect

This review summarises recent advances in quantum chemical calculations of base-stacking forces in nucleic acids. We explain in detail the very complex relationship between the gas-phase basestacking energies, as revealed by quantum chemical (QM) calculations, and the highly variable roles of these interactions in nucleic acids. This issue is rarely discussed in quantum chemical and physical chemistry literature. We further extensively discuss methods that are available for basestacking studies, complexity of comparison of stacking calculations with gas phase experiments, balance of forces in stacked complexes of nucleic acid bases, and the relation between QM and force field descriptions. We also review all recent calculations on base-stacking systems, including details analysis of the B-DNA stacking. Specific attention is paid to the highest accuracy QM calculations, to the decomposition of the interactions, and development of dispersion-balanced DFT methods. Future prospects of computational studies of base stacking are discussed.

Sponer, Jiri; Riley, Kevin E.; Hobza, Pavel

2008-04-07

187

Nucleic acid-protein interactions: Wedding for love or circumstances?  

PubMed

The sixth Figeac meeting on nucleic acid-protein interactions was held in Figeac, France, from September 26th to October 1st, 2008. It was organized by the working group "nucleic acid-protein interactions and gene expression" from the French Society for Biochemistry and Molecular Biology. This report briefly summarizes the presentations by 40 speakers during the four plenary sessions, which were organised as follows: (1) nucleic acids: targets and tools, (2) RNA superstar, (3) nuclear structure and dynamics, and (4) new concepts - new approaches. A total of 22 plenary lectures, 18 oral communications and 40 posters were presented over the 5 days, providing a highly stimulating environment for scientific exchange between the approximately 80 participants (biochemists, physicists, bio-informaticians and molecular and cellular biologists). PMID:19422875

Lavelle, Christophe; Buckle, Malcolm

2009-08-01

188

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.

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

2013-01-01

189

Amino-containing magnetic nanoemulsions: elaboration and nucleic acid extraction  

NASA Astrophysics Data System (ADS)

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

Veyret, Raphael; Delair, Thierry; Pichot, Christian; Elaissari, Abdelhamid

2005-08-01

190

Circulating nucleic acids as biomarkers in breast cancer  

PubMed Central

During tumor development, tumor cells release their nucleic acids into the blood circulation. This process occurs by apoptotic and necrotic cell deaths along with active cell secretion, resulting in high levels of circulating DNA, mRNA, and microRNA in the blood of patients with breast cancer. As circulating cell-free tumor nucleic acids may reflect the characteristics of the primary tumor and even of micrometastatic cells, they may be excellent blood biomarkers for screening breast cancer. Assays that allow the repetitive monitoring of patients by using blood samples as liquid biopsy may be efficient in assessing cancer progression in patients whose tumor tissue is not available. This review evaluates the recent data on the potential use of circulating cell-free nucleic acids as biomarkers for breast cancer.

2013-01-01

191

Molecular properties and medical applications of Peptide nucleic acids.  

PubMed

Peptide Nucleic Acids (PNAs) are molecules combining structural features of proteins and nucleic acid. They resemble DNA or RNA by forming helical polyamides containing nitrogen bases attached to the backbone consisting of N-(2-aminoethyl)-glycine monomers, which mimics the alternating ribose-phosphodiester-backbone of a nucleic acid. Because PNAs bind exceptionally strong to complementary DNA or RNA sequences obeying Watson-Crick base paring, they became attractive candidates for antisense and antigen therapies. PNAs are also being tested as novel antibiotics, gene-activating agents, and as molecular probes for FISH and imaging or biosensors used in diagnostics. Although PNAs offer many exiting medical applications, improving their cellular uptake and developing specific delivery strategies is crucial for a successful entry in the clinic in the near future. PMID:24766383

Malcher, Jakub; Wesoly, Joanna; Bluyssen, Hans A R

2014-05-01

192

Nucleic acid detection technologies and marker molecules in bacterial diagnostics.  

PubMed

There is a growing need for quick and reliable methods for microorganism detection and identification worldwide. Although traditional culture-based technologies are trustworthy and accurate at a relatively low cost, they are also time- and labor-consuming and are limited to culturable bacteria. Those weaknesses have created a necessity for alternative technologies that are capable for faster and more precise bacterial identification from medical, food or environmental samples. The most common current approach is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to the specific nucleic acid profiles that are present. This review aims to give an up-to-date overview of different nucleic acid target sequences and respective analytical technologies. PMID:24724586

Scheler, Ott; Glynn, Barry; Kurg, Ants

2014-05-01

193

Absence of parallelism between polyamine and nucleic acid contents during induced growth of cucumber cotyledons.  

PubMed Central

The cytokinins (benzyladenine or benzyladenosine) decreased spermidine and spermine contents despite increasing putrescine content, when administered to isolated cotyledons of Cucumis sativus L. var. Guntur in organ culture. KCl decreased putrescine contents, although marginally increasing polyamine contents. The cytokinins and/or KCl augmented nucleic acid biosynthesis and accumulation, resulting in enhanced growth and differentiation of the isolated cotyledons. These observations show that polyamine accumulation and growth are not always coupled.

Suresh, M R; Adiga, P R

1978-01-01

194

Kit for detecting nucleic acid sequences using competitive hybridization probes  

DOEpatents

A kit is provided for detecting a target nucleic acid sequence in a sample, the kit comprising: a first hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the first hybridization probe including a first complexing agent for forming a binding pair with a second complexing agent; and a second hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the first hybridization probe does not selectively hybridize, the second hybridization probe including a detectable marker; a third hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the third hybridization probe including the same detectable marker as the second hybridization probe; and a fourth hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the third hybridization probe does not selectively hybridize, the fourth hybridization probe including the first complexing agent for forming a binding pair with the second complexing agent; wherein the first and second hybridization probes are capable of simultaneously hybridizing to the target sequence and the third and fourth hybridization probes are capable of simultaneously hybridizing to the target sequence, the detectable marker is not present on the first or fourth hybridization probes and the first, second, third, and fourth hybridization probes each include a competitive nucleic acid sequence which is sufficiently complementary to a third portion of the target sequence that the competitive sequences of the first, second, third, and fourth hybridization probes compete with each other to hybridize to the third portion of the target sequence.

Lucas, Joe N. (San Ramon, CA); Straume, Tore (Tracy, CA); Bogen, Kenneth T. (Walnut Creek, CA)

2001-01-01

195

Recent Advances in Chemical Modification of Peptide Nucleic Acids  

PubMed Central

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

Rozners, Eriks

2012-01-01

196

Nanopores and nucleic acids: prospects for ultrarapid sequencing  

NASA Technical Reports Server (NTRS)

DNA and RNA molecules can be detected as they are driven through a nanopore by an applied electric field at rates ranging from several hundred microseconds to a few milliseconds per molecule. The nanopore can rapidly discriminate between pyrimidine and purine segments along a single-stranded nucleic acid molecule. Nanopore detection and characterization of single molecules represents a new method for directly reading information encoded in linear polymers. If single-nucleotide resolution can be achieved, it is possible that nucleic acid sequences can be determined at rates exceeding a thousand bases per second.

Deamer, D. W.; Akeson, M.

2000-01-01

197

Biomimetic high density lipoprotein nanoparticles for nucleic acid delivery.  

PubMed

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

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

2011-03-01

198

Biomimetic High Density Lipoprotein Nanoparticles For Nucleic Acid Delivery  

PubMed Central

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

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

2014-01-01

199

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.

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

2011-01-01

200

Altered nucleic acid partitioning during phenol extraction or silica adsorption by guanidinium and potassium salts  

Microsoft Academic Search

Nucleic acids were found to partition into the phenol phase during phenol extraction in the presence of guanidinium at certain concentrations under acidic conditions. The guanidinium-concentration-dependent nucleic acid partitioning patterns were analogous to those of the nucleic acid adsorption\\/partitioning onto silica mediated by guanidinium, which implied that phenol and silica interact with nucleic acids through similar mechanisms. A competition effect

Lei Xu; Jun Lv; Liefeng Ling; Peng Wang; Ping Song; Ruirui Su; Guoping Zhu

2011-01-01

201

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

202

Selective Attachment of Nucleic Acid Molecules to Patterned Self-Assembled Surfaces.  

National Technical Information Service (NTIS)

Patterns of pre-formed hybridizable nucleic acid oligomers are formed upon a substrate. The substrate is coated with molecules, such as aminosilanes, whose reactivity with nucleic acid molecules can be transformed by irradiation. The coated substrate expo...

L. A. Chrisey W. J. Dressick J. M. Calvert

1994-01-01

203

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

204

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.

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

205

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.

Storck, Roger

1965-01-01

206

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

207

Gene delivery by a steroid-peptide nucleic acid conjugate  

Microsoft Academic Search

We previously introduced a method called steroid-mediated gene delivery (SMGD), which uses steroid receptors as shuttles to facilitate the nuclear uptake of transfected DNA. Here, we describe a SMGD strategy with peptide nucleic acids (PNAs) that allowed linkage of a steroid molecule to a defined position in a plasmid without disturbing its gene expression. We synthesized and tested several bifunctional

Alexandre G. Rebuffat; Andrea R. Nawrocki; Peter E. Nielsen; Alessio G. Bernasconi; Eloy Bernal-Mendez; Brigitte M. Frey; Felix J. Frey

2002-01-01

208

Advances in fluorescent tracking of nucleic acids in living cells  

Microsoft Academic Search

Nucleic acids are typically detected in morphologically preserved fixed cells and tissues using in situ hybridization techniques. This review discusses a variety of established and more challenging fluorescence-based methods for the detection and tracking of DNA or RNA sequences in living cells. Over the past few years, various fluorescent in vivo labeling methods have been developed, and dedicated microscope and

Roeland W. Dirks; Hans J. Tanke

2006-01-01

209

Arrays of nucleic acid probes on biological chips  

DOEpatents

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

Chee, Mark (Palo Alto, CA); Cronin, Maureen T. (Los Altos, CA); Fodor, Stephen P. A. (Palo Alto, CA); Huang, Xiaohua X. (Mt. View, CA); Hubbell, Earl A. (Mt. View, CA); Lipshutz, Robert J. (Palo Alto, CA); Lobban, Peter E. (Palo Alto, CA); Morris, MacDonald S. (San Jose, CA); Sheldon, Edward L. (Menlo Park, CA)

1998-11-17

210

Universal eubacteria nucleic acid probes and assay methods  

US Patent & Trademark Office Database

Nucleic acid probes capable of hybridizing to rRNA of eubacteria and not to rRNA of non-eubacteria are described along with methods utilizing such probes for the detection of eubacteria in clinical and other samples. Preferred embodiments include probes capable of distinguishing between gram-positive and gram-negative bacteria.

1995-03-28

211

Enhanced oligonucleotide-mediated nucleic acid sequence alteration  

US Patent & Trademark Office Database

The invention is directed to oligonucleotide-mediated repair or alteration of genetic information, such as nucleic acid sequence alteration, and methods, compositions and kits for enhancing the efficiency of such alteration. Specifically, the invention incorporates the use of factors such as Histone Deacetylase Inhibitor (HDAC), Lambda phage beta protein, or hydroxyurea to achieve such enhanced efficiency.

2009-07-28

212

Nucleic acid probes in diagnosis of viral diseases of man  

Microsoft Academic Search

Summary With the recent, rapid advances in recombinant DNA technology, it has become possible to consider the use of nucleic acid probes in diagnosis of human viral diseases. Several examples are discussed which employ techniques of dot blot hybridization, sandwich hybridization andin situ hybridization. Typing of viral strains using restriction endonuclease digestion as an epidemiological tool is considered. Finally, the

J. K. Kulski; Mary Norval

1985-01-01

213

THE BINDING OF VIOLAMYCIN TO DOUBLE STRAMDED NUCLEIC ACIDS  

Microsoft Academic Search

SUMMARY We studied the binding of violamycin BI to some double stranded synthetic nucleic acids. We evidenced the formation of stacked complex on the polymer surface at very low p (ratio of polymer to ligand) and determined the binding parameters using Schwarz method. At high values of p it forms the intercalation complex between the base pairs and used Scatchard

Tatiana Oncescu; Simona Radulescu

214

Biorelevant Latexes and Microgels for the Interaction with Nucleic Acids  

Microsoft Academic Search

This chapter is a review of recent work devoted to polymer colloids with nucleic acids in the domain of biomedical diagnostic, in which latex particles are used as carrier. After a brief introduction concerning the applications of latex particles in the biomedical field, the first part describes the routes leading to the elaboration of reactive latexes using radical-initiated polymerization in

Abdelhamid Elaissari; François Ganachaud; Christian Pichot

215

Sandwich probes: two simultaneous reactions for templated nucleic acid detection.  

PubMed

Fluorescence-quenched nucleic acid probes with reactive moieties at both the 5' and 3' ends are synthesized and tested for reaction with two adjacent nucleophile-containing DNAs. These probes improve signal to background over singly reactive probes and can discriminate single nucleotide polymorphisms in the target DNA or RNA. PMID:20927470

Kleinbaum, Daniel J; Kool, Eric T

2010-11-21

216

A DNA origami nanorobot controlled by nucleic Acid hybridization.  

PubMed

A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators. PMID:24648163

Torelli, Emanuela; Marini, Monica; Palmano, Sabrina; Piantanida, Luca; Polano, Cesare; Scarpellini, Alice; Lazzarino, Marco; Firrao, Giuseppe

2014-07-01

217

Application of Controlled Radical Polymerization for Nucleic Acid Delivery  

PubMed Central

CONSPECTUS Nucleic acid-based therapeutics can potentially address otherwise untreatable genetic disorders and have significant potential for a wide range of diseases. Therapeutic gene delivery can restore protein function by replacing defunct genes to restore cellular health while RNA interference (RNAi) can mask mutated and harmful genes. Cationic polymers have been extensively studied for nucleic acid delivery applications due to their self-assembly with nucleic acids into virus-sized nanoparticles and high transfection efficiency in vitro, but toxicity and particle stability have limited their clinical applications. The advent of controlled radical polymerization has improved the quality, control and reproducibility of synthesized materials. Controlled radical polymerization yields well-defined, narrowly disperse materials of designable architectures and molecular weight, allowing study of the effects of polymer architecture and molecular weight on transfection efficiency and cytotoxicity for improved design of next-generation vectors. Robust methods such as atom transfer radical polymerization (ATRP), reverse addition-fragmentation chain transfer polymerization (RAFT), and ring-opening metastasis polymerization (ROMP) have been used to engineer materials that specifically enhance extracellular stability, cellular specificity, and decrease toxicity. This Account reviews findings from structure-function studies that have elucidated key design motifs necessary for the development of effective nucleic acid vectors. In addition, polymers that are biodegradable, form supramolecular structures, target specific cells, or facilitate endosomal release are also discussed. Finally, promising materials with in vivo applications ranging from pulmonary gene delivery to DNA vaccines are described.

CHU, DAVID S.H.; SCHELLINGER, JOAN G.; SHI, JULIE; CONVERTINE, ANTHONY J.; STAYTON, PATRICK S.; PUN, SUZIE H.

2012-01-01

218

Does the Agent of Scrapie Replicate without Nucleic Acid ?  

Microsoft Academic Search

Scrapie is a slowly developing disease of the nervous system. Experiments on the effects of ultra-violet irradiation of suspensions of infected mouse brain extracts confirm that the agent responsible for it does not depend on a nucleic acid for its ability to replicate. No evidence is obtained, however, to indicate whether the agent is associated with a protein.

Tikvah Alper; W. A. Cramp; D. A. HAIG; M. C. CLARKE

1967-01-01

219

Mfold web server for nucleic acid folding and hybridization prediction  

Microsoft Academic Search

The abbreviated name,'mfold web server',describes a number of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of

Michael Zuker

2003-01-01

220

Microfluidic sample preparation: cell lysis and nucleic acid purification  

Microsoft Academic Search

Due to the lack of development in the area of sample preparation, few complete lab-on-a-chip systems have appeared in recent years that can deal with raw samples. Cell lysis and nucleic acid extraction systems are sufficiently complex even before adding the complexity of an analysis system. In this review, a variety of microfluidic sample preparation methods are discussed and evaluated.

Jungkyu Kim; Michael Johnson; Parker Hill; Bruce K. Gale

2009-01-01

221

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

222

Crystallographic Studies of Chemically Modified Nucleic Acids: A Backward Glance  

PubMed Central

Chemically modified nucleic acids (CNAs) are widely explored as antisense oligonucleotide or small interfering RNA (siRNA) candidates for therapeutic applications. CNAs are also of interest in diagnostics, high-throughput genomics and target validation, nanotechnology and as model systems in investigations directed at a better understanding of the etiology of nucleic acid structure as well as the physical-chemical and pairing properties of DNA and RNA and for probing protein-nucleic acid interactions. In this article we review research conducted by our laboratory over the past two decades with a focus on crystal structure analyses of CNAs and artificial pairing systems. We highlight key insights into issues ranging from conformational distortions as a consequence of modification to the modulation of pairing strength and RNA affinity by stereoelectronic effects and hydration. Although crystal structures have only been determined for a subset of the large number of modifications that were synthesized and analyzed in the oligonucleotide context to date, they have yielded guiding principles for the design of new analogs with tailormade properties, including pairing specificity, nuclease resistance and cellular uptake. And, perhaps less obviously, crystallographic studies of CNAs and synthetic pairing systems have shed light on fundamental aspects of DNA and RNA structure and function that would not have been disclosed by investigations solely focused on the natural nucleic acids.

Egli, Martin; Pallan, Pradeep S.

2010-01-01

223

Nucleic acids encoding human trithorax protein  

DOEpatents

In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

Evans, Glen A. (Encinitas, CA); Djabali, Malek (Marseilles, FR); Selleri, Licia (Del Mar, CA); Parry, Pauline (San Diego, CA)

2001-01-01

224

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

Federal Register 2010, 2011, 2012, 2013

...proposing to reclassify nucleic acid-based in vitro diagnostic devices for the detection of...Controls Guideline: Nucleic Acid-Based In Vitro Diagnostic Devices for the Detection of...of the Device A nucleic acid-based in vitro diagnostic device for the detection...

2013-06-19

225

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

Federal Register 2010, 2011, 2012, 2013

...proposing to reclassify nucleic acid-based in vitro diagnostic devices for the detection of...Background of the Device Nucleic acid-based in vitro diagnostic devices for the detection of...Identification Nucleic acid-based in vitro diagnostic devices for the detection...

2012-03-19

226

Thermodynamic database for protein-nucleic acid interactions (ProNIT)  

Microsoft Academic Search

Motivation: Protein-nucleic acid interactions are funda- mental to the regulation of gene expression. In order to elucidate the molecular mechanism of protein-nucleic acid recognition and analyze the gene regulation network, not only structural data but also quantitative binding data are necessary. Although there are structural databases for proteins and nucleic acids, there exists no database for their experimental binding data.

Ponraj Prabakaran; Jianghong An; M. Michael Gromiha; Samuel Selvaraj; Hatsuho Uedaira; Hidetoshi Kono; Akinori Sarai

2001-01-01

227

The use of solid supports to generate nucleic acid carriers.  

PubMed

Nucleic acids are the foundation stone of all cellular processes. Consequently, the use of DNA or RNA to treat genetic and acquired disorders (so called gene therapy) offers enormous potential benefits. The restitution of defective genes or the suppression of malignant genes could target a range of diseases, including cancers, inherited diseases (cystic fibrosis, muscular dystrophy, etc.), and viral infections. However, this strategy has a major barrier: the size and charge of nucleic acids largely restricts their transit into eukaryotic cells. Potential strategies to solve this problem include the use of a variety of natural and synthetic nucleic acid carriers. Driven by the aim and ambition of translating this promising therapeutic approach into the clinic, researchers have been actively developing advanced delivery systems for nucleic acids for more than 20 years. A decade ago we began our investigations of solid-phase techniques to construct families of novel nucleic acid carriers for transfection. We envisaged that the solid-phase synthesis of polycationic dendrimers and derivatized polyamimes would offer distinct advantages over solution phase techniques. Notably in solid phase synthesis we could take advantage of mass action and streamlined purification procedures, while simplifying the handling of compounds with high polarities and plurality of functional groups. Parallel synthesis methods would also allow rapid access to libraries of compounds with improved purities and yields over comparable solution methodologies and facilitate the development of structure activity relationships. We also twisted the concept of the solid-phase support on its head: we devised miniaturized solid supports that provided an innovative cell delivery vehicle in their own right, carrying covalently conjugated cargos (biomolecules) into cells. In this Account, we summarize the main outcomes of this series of chemically related projects. PMID:22390230

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

2012-07-17

228

NAFlex: a web server for the study of nucleic acid flexibility.  

PubMed

We present NAFlex, a new web tool to study the flexibility of nucleic acids, either isolated or bound to other molecules. The server allows the user to incorporate structures from protein data banks, completing gaps and removing structural inconsistencies. It is also possible to define canonical (average or sequence-adapted) nucleic acid structures using a variety of predefined internal libraries, as well to create specific nucleic acid conformations from the sequence. The server offers a variety of methods to explore nucleic acid flexibility, such as a colorless wormlike-chain model, a base-pair resolution mesoscopic model and atomistic molecular dynamics simulations with a wide variety of protocols and force fields. The trajectories obtained by simulations, or imported externally, can be visualized and analyzed using a large number of tools, including standard Cartesian analysis, essential dynamics, helical analysis, local and global stiffness, energy decomposition, principal components and in silico NMR spectra. The server is accessible free of charge from the mmb.irbbarcelona.org/NAFlex webpage. PMID:23685436

Hospital, Adam; Faustino, Ignacio; Collepardo-Guevara, Rosana; González, Carlos; Gelpí, Josep Lluis; Orozco, Modesto

2013-07-01

229

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.

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

230

Method for promoting specific alignment of short oligonucleotides on nucleic acids  

DOEpatents

Disclosed is a method for promoting specific alignment of short oligonucleotides on a nucleic acid polymer. The nucleic acid polymer is incubated in a solution containing a single-stranded DNA-binding protein and a plurality of oligonucleotides which are perfectly complementary to distinct but adjacent regions of a predetermined contiguous nucleotide sequence in the nucleic acid polymer. The plurality of oligonucleotides anneal to the nucleic acid polymer to form a contiguous region of double stranded nucleic acid. Specific application of the methods disclosed include priming DNA synthesis and template-directed ligation.

Studier, F. William (Stony Brook, NY); Kieleczawa, Jan (Coram, NY); Dunn, John J. (Bellport, NY)

1996-01-01

231

Recent Developments in Peptide-Based Nucleic Acid Delivery  

PubMed Central

Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cell-penetrating peptides (CPPs). The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10–30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisense-oligonucleotides), which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls.

Veldhoen, Sandra; Laufer, Sandra D.; Restle, Tobias

2008-01-01

232

Use of acid, alkali and enzymes for the extraction of nucleic acids from pollen  

Microsoft Academic Search

Various methods for extracting nucleic acids from pollen were tested to find a suitable procedure for obtaining a pure preparation\\u000a of nucleic acids uncontaminated by polysaccharides and polyphosphates without the use of ion exchangers. Extraction was carried\\u000a out with perchloric acid, potassium hydroxide, ribonuclease and deoxyribonuclease, sodium tetraborate, and combinations of\\u000a these. In all fractions, residues of precipitates and residues

J. Süss

1970-01-01

233

Green technologies for room temperature nucleic acid storage.  

PubMed

Maintaining the long-term integrity of nucleic acids in the laboratory has traditionally required the use of freezers. However, novel nucleic acid stabilization technologies may allow for the storage of DNA and RNA at room temperature in a cost-effective, environmentally friendly manner. In this study, we evaluated two novel products for room temperature DNA storage: Biomatrica's DNA SampleMatrix technology and GenVault's GenTegra DNA technology. We compared the integrity and quality of DNA stored using these products against DNA stored in a -20 C freezer by performing downstream testing with short range PCR, long range PCR, DNA sequencing, and SNP microarrays. In addition, we tested Biomatrica's RNAstable product for its ability to preserve RNA at room temperature for use in a quantitative reverse transcription PCR assay. PMID:19801719

Wan, Eunice; Akana, Matthew; Pons, Jennifer; Chen, Justin; Musone, Stacy; Kwok, Pui-Yan; Liao, Wilson

2010-01-01

234

Fool's Gold Footprinting: microfluidic probing of nucleic acids  

NASA Astrophysics Data System (ADS)

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

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

2012-02-01

235

Phase transitions in sequence matches and nucleic acid structure.  

PubMed

Analyses of phase transitions in biopolymers have previously been restricted to studies of average behavior along macromolecules. Extremal properties, such as longest helical region, can now be studied with a new family of probability distributions [Arratia, R., Gordon, L. & Waterman, M. S. (1986) Ann. Stat. 14, 971-993]. Not only is such extremal behavior analyzed with great precision, but new phase transitions are determined. One phase transition occurs when behavior of the free energy of the longest helical region abruptly changes from proportional to sequence length. The annealing of two single-stranded molecules and the melting of a double helix are both considered. These results, initially suggested by studies of optimal matching of random DNA sequences [Smith, T. F., Waterman, M. S. & Burks, C. (1985) Nucleic Acids Res. 13, 645-656], also have importance for significance tests in comparison of nucleic acid or protein sequences. PMID:3469666

Waterman, M S; Gordon, L; Arratia, R

1987-03-01

236

Phase Transitions in Sequence Matches and Nucleic Acid Structure  

NASA Astrophysics Data System (ADS)

Analyses of phase transitions in biopolymers have previously been restricted to studies of average behavior along macromolecules. Extremal properties, such as longest helical region, can now be studied with a new family of probability distributions [Arratia, R., Gordon, L. & Waterman, M. S. (1986) Ann. Stat. 14, 971-993]. Not only is such extremal behavior analyzed with great precision, but new phase transitions are determined. One phase transition occurs when behavior of the free energy of the longest helical region abruptly changes from proportional to logarithm of the sequence length to proportional to sequence length. The annealing of two single-stranded molecules and the melting of a double helix are both considered. These results, initially suggested by studies of optimal matching of random DNA sequences [Smith, T. F., Waterman, M. S. & Burks, C. (1985) Nucleic Acids Res. 13, 645-656], also have importance for significance tests in comparison of nucleic acid or protein sequences.

Waterman, Michael S.; Gordon, Louis; Arratia, Richard

1987-03-01

237

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

238

Nucleic Acid Aptamers: Research Tools in Disease Diagnostics and Therapeutics  

PubMed Central

Aptamers are short sequences of nucleic acid (DNA or RNA) or peptide molecules which adopt a conformation and bind cognate ligands with high affinity and specificity in a manner akin to antibody-antigen interactions. It has been globally acknowledged that aptamers promise a plethora of diagnostic and therapeutic applications. Although use of nucleic acid aptamers as targeted therapeutics or mediators of targeted drug delivery is a relatively new avenue of research, one aptamer-based drug “Macugen” is FDA approved and a series of aptamer-based drugs are in clinical pipelines. The present review discusses the aspects of design, unique properties, applications, and development of different aptamers to aid in cancer diagnosis, prevention, and/or treatment under defined conditions.

Yadava, Pramod K.

2014-01-01

239

Green Technologies for Room Temperature Nucleic Acid Storage  

PubMed Central

Maintaining the long-term integrity of nucleic acids in the laboratory has traditionally required the use of freezers. However, novel nucleic acid stabilization technologies may allow for the storage of DNA and RNA at room temperature in a cost-effective, environmentally friendly manner. In this study, we evaluated two novel products for room temperature DNA storage: Biomatrica’s DNA SampleMatrix technology and GenVault’s GenTegra DNA technology. We compared the integrity and quality of DNA stored using these products against DNA stored in a ?20°C freezer by performing downstream testing with short range PCR, long range PCR, DNA sequencing, and SNP microarrays. In addition, we tested Biomatrica’s RNAstable product for its ability to preserve RNA at room temperature for use in a quantitative reverse transcription PCR assay.

Wan, Eunice; Akana, Matthew; Pons, Jennifer; Chen, Justin; Musone, Stacy; Kwok, Pui-Yan; Liao, Wilson

2014-01-01

240

Lipid nanoparticles as vehicles for macromolecules: nucleic acids and peptides.  

PubMed

Traditional drug delivery systems are not efficient for peptide, protein and nucleic acid (plasmid DNA, oligonucleotides or short interfering RNA) delivery, thereby LNP have been exploited as potential delivery and targeting systems of these molecules. Since their discovery in the early 90's several research groups have focused their efforts on the improvement of this kind of nanocarriers in terms of effectiveness and safety. This review features the recent and most relevant patents related to these topics, with particular attention to targeting and protection from environmental agents. Moreover, in the case of nucleic acids strategies to improve transfection mediated by lipid nanoparticles (entrance to the cells, intracellular distribution or going through nuclear envelope) will be assessed. Regarding peptides and proteins, enhancement of encapsulation efficiency and absorption through mucoses are the main studied drawbacks. Finally, this work also includes a summary of the existing patents about the use of LNP as immune response adjuvants by using either plasmid DNA or proteins. PMID:21834776

del Pozo-Rodríguez, Ana; Delgado, Diego; Solinís, Maria A; Gascón, Alicia R

2011-09-01

241

Biological activity and biotechnological aspects of locked nucleic acids.  

PubMed

Locked nucleic acid (LNA) is one of the most promising new nucleic acid analogues that has been produced under the past two decades. In this chapter, we have tried to cover many of the different areas, where this molecule has been used to improve the function of synthetic oligonucleotides (ONs). The use of LNA in antisense ONs, including gapmers, splice-switching ONs, and siLNA, as well as antigene ONs, is reviewed. Pharmacokinetics as well as pharmacodynamics of LNA ONs and a description of selected compounds in, or close to, clinical testing are described. In addition, new LNA modifications and the adaptation of enzymes for LNA incorporation are reviewed. Such enzymes may become important for the development of stabilized LNA-containing aptamers. PMID:23721720

Lundin, Karin E; Højland, Torben; Hansen, Bo R; Persson, Robert; Bramsen, Jesper B; Kjems, Jørgen; Koch, Troels; Wengel, Jesper; Smith, C I Edvard

2013-01-01

242

Reactions between Singlet Oxygen and the Constituents of Nucleic Acids  

PubMed Central

Bases, nucleosides, nucleotides, and polynucleotides were exposed to chemically generated singlet oxygen to determine whether the species oxidized paralleled those oxidized in photodynamic reactions. In neutral or basic aqueous solution guanine, guanosine, deoxyguanosine, guanylic acid, deoxyguanylic acid, thymine, and uracil reacted with singlet oxygen. Since these compounds are oxidized in photodynamic processes, this study provides further evidence that singlet oxygen is the active intermediate in the photodynamic oxidation of nucleic acid constituents. Dienophilic attack by singlet oxygen is considered to be a plausible mechanism in these reactions.

Hallett, F. R.; Hallett, B. P.; Snipes, W.

1970-01-01

243

Method and apparatus for staining immobilized nucleic acids  

SciTech Connect

A method for staining immobilized nucleic acids includes the following steps: affixing DNA probes to a solid substrate; moving target DNA material into proximity with the DNA probes, whereby the target DNA hybridized with specific ones of the DNA probes; and moving a fluorescent dye into proximity with the hybridized target DNA, whereby the fluorescent dye binds to the hybridized DNA to enable subsequent detection of fluorescence.

Ramsey, J.M.; Foote, R.S.; Jacobson, S.C.

2000-05-02

244

Exploring nucleic acid modifications in RNA hairpins and antisense oligonucleotides  

Microsoft Academic Search

Over 100 different natural nucleic acid modifications are present in nature. In the first part of this thesis work (Chapters 1--4), the focus is directed on studying the most common type of modification, pseudouridine. Pseudouridine has been observed to be confined in areas of functional importance. For example, the RNA hairpin helix 69 (H69) of Escherichia coli 23S rRNA contains

Jean-Paul Desaulniers

2005-01-01

245

Interfacial nucleic acid chemistry studied by acoustic shear wave propagation  

Microsoft Academic Search

Acoustic wave devices have continued to gain attention as biosensor structures because of their relative ease of operation and sensitivity to interfacial biochemical events. In the present paper, we review the use of the thickness-shear mode device for the label-free detection of processes involving nucleic acid moieties that are imposed at the sensor–liquid interface. Following a concise discussion of the

Biljana A Cavic; Michael Thompson

2002-01-01

246

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

247

Nucleic acid-based tissue biomarkers of urologic malignancies.  

PubMed

Abstract 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

248

Principles of Nucleic Acid Cleavage by Metal Ions  

Microsoft Academic Search

\\u000a Nucleic acids are responsible for the storage, transmission, and expression of genetic information in all living organisms.\\u000a These macromolecules tend to behave as salts in solution since the negative charges of the polynucleotide phosphodiester backbone\\u000a are usually neutralized by metal solute ions. Therefore, for practical purposes, it is difficult if not impossible to separate\\u000a the behavior of DNA and RNA

A. Dallas; A. V. Vlassov; S. A. Kazakov

249

Developing nucleic acid-based electrical detection systems  

PubMed Central

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

Gabig-Ciminska, Magdalena

2006-01-01

250

Immune Recognition of Nucleic Acids and Their Metabolites  

Microsoft Academic Search

\\u000a Recent research suggests that nucleic acids are active modulators of the immune system. RNA and DNA can be detected by specific\\u000a receptors – the so-called Toll-like receptors, RIG-I-like receptors, and NOD-like receptors. Resultant intra- and intercellular\\u000a activations of the innate immune system are pivotal in both protective and pathological immune responses during infection\\u000a and other immunological disorders. Moreover, our immune

Shohei Koyama; Shizuo Akira; Ken J. Ishii

251

Biorelevant Latexes and Microgels for the Interaction with Nucleic Acids  

Microsoft Academic Search

This chapter is a review of recent work devoted to polymer colloids with nucleic acids in the domain of biomedical diagnostic,\\u000a in which latex particles are used as carrier. After a brief introduction concerning the applications of latex particles in\\u000a the biomedical field, the first part describes the routes leading to the elaboration of reactive latexes using radical-initiated\\u000a polymerization in

Abdelhamid Elaissari; François Ganachaud; Christian Pichot

252

Method and apparatus for staining immobilized nucleic acids  

DOEpatents

A method for staining immobilized nucleic acids includes the steps of affixing DNA probes to a solid substrate, moving target DNA material into proximity with the DNA probes, whereby the target DNA hybridized with specific ones of the DNA probes, and moving a fluorescent dye into proximity with the hybridized target DNA, whereby the fluorescent dye binds to the hybridized DNA to enable subsequent detection of fluorescence.

Ramsey, J. Michael (Knoxville, TN); Foote, Robert S. (Oak Ridge, TN); Jacobson, Stephen C. (Knoxville, TN)

2000-01-01

253

NMR solution structures of LNA (locked nucleic acid) modified quadruplexes  

Microsoft Academic Search

We have determined the NMR solution structures of the quadruplexes formed by d(TGLGLT) and d(TL4T), where L denotes LNA (locked nucleic acid) modified G-residues. Both structures are tetrameric, parallel and right-handed and the native global fold of the corresponding DNA quadruplex is retained upon introduction of the LNA nucleotides. However, local structural alterations are observed owing to the locked LNA

Jakob T. Nielsen; Khalil Arar; Michael Petersen

2006-01-01

254

Design of antisense oligonucleotides stabilized by locked nucleic acids  

Microsoft Academic Search

The design of antisense oligonucleotides containing locked nucleic acids (LNA) was optimized and compared to intensively studied DNA oligonucleotides, phosphorothioates and 2'-O-methyl gapmers. In contradiction to the literature, a stretch of seven or eight DNA monomers in the center of a chimeric DNA\\/LNA oligonucleotide is necessary for full activation of RNase H to cleave the target RNA. For 2'-O-methyl gapmers

Jens Kurreck; Eliza Wyszko; Clemens Gillen; Volker A. Erdmann

2002-01-01

255

Prospects for antisense peptide nucleic acid (PNA) therapies for HIV  

PubMed Central

Since the discovery and synthesis of a novel DNA mimic, peptide nucleic acid (PNA) in 1991, PNAs have attracted tremendous interest and have shown great promise as potential antisense drugs. They have been used extensively as tools for specific modulation of genes expression by targeting translation or transcription processes. This review discusses the present and future therapeutic potential of this class of compound as anti-HIV-1 drugs.

Pandey, Virendra N.; Upadhyay, Alok; Chaubey, Binay

2009-01-01

256

Cell-free Nucleic Acids as Potential Markers for Preeclampsia  

Microsoft Academic Search

Preeclampsia is one of the leading causes of maternal and fetal\\/neonatal mortality and morbidity worldwide. Therefore, widely applicable and affordable tests are needed to make an early diagnosis before the occurrence of the clinical symptoms. Circulating cell-free nucleic acids in plasma and serum are novel biomarkers with promising clinical applications in different medical fields, including prenatal diagnosis.Quantitative changes of cell-free

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

2011-01-01

257

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

258

Non-natural Nucleic Acids for Synthetic Biology  

PubMed Central

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

Appella, Daniel H.

2011-01-01

259

Nucleic acid hybridization using DNA covalently coupled to cellulose.  

PubMed

We describe a method for linking RNA and DNA covalently to finely divided cellulose through a diazotized aryl amine, which reacts primarily with guanine and uracil (thymine) residues of single strands. The high efficiency of coupling and high capacity of the cellulose for nucleic acid make possible a product with as much as 67 mug of nucleic acid per mg of cellulose. The product is especially suitable for hybridization experiments where very low backgrounds are important, and it is stable in 99% formamide at 80 degrees C so that hybridized nucleic acid can be recovered easily. Full length linear Simian Virus 40 (SV40) DNA, produced by cleavage of SV40(I) DNA with S1 nuclease, can be coupled to diazo cellulose with an efficiency of 80-90%, and is effective in hybridization experiments with SV40 DNA, complementary RNA synthesized in vitro from SV40(I) DNA with E. coli RNA polymerase, and the SV40-specific fraction of total RNA from SV40-infected and transformed cells. In these experiments an excess of cellulose-bound DNA was used, and the efficiency of hybridization was about 90% when ribonuclease treatment of the hybrids was omitted. PMID:167982

Noyes, B E; Stark, G R

1975-07-01

260

Identification and Characterization of a Cell Membrane Nucleic Acid Channel  

NASA Astrophysics Data System (ADS)

We have identified a 45-kDa protein purified from rat renal brush border membrane that binds short single-stranded nucleic acid sequences. This activity was purified, reconstituted in proteoliposomes, and then fused with model planar lipid bilayers. In voltage-clamp experiments, the reconstituted 45-kDa protein functioned as a gated channel that allows the passage of nucleic acids. Channel activity was observed immediately after addition of oligonucleotide. Channel activity was not observed in the absence of purified protein or of oligonucleotide or when protein was heat-inactivated prior to forming proteoliposomes. In the presence of symmetrical buffered solution and oligonucleotide, current passed linearly over the range of holding potentials tested. Conductance was 10.4 ± 0.4 picosiemens (pS) and reversal potential was 0.2 ± 1.7 mV. There was no difference in channel conductance or reversal potential between phosphodiester and phosphorothioate oligonucleotides. Ionsubstitution experiments documented a shift in reversal potential only when a concentration gradient for oligonucleotide was established, indicating that movement of oligonucleotide alone was responsible for current. Movement of oligonucleotide across the bilayer was confirmed by using 32P-labeled oligonucleotides. Channel open probability decreased significantly in the presence of heparan sulfate. These studies provide evidence for a cell surface channel that conducts nucleic acids.

Hanss, Basil; Leal-Pinto, Edgar; Bruggeman, Leslie A.; Copeland, Terry D.; Klotman, Paul E.

1998-02-01

261

A Computational Approach to Modeling Nucleic Acid Hairpin Structures  

PubMed Central

Hairpin is a structural motif frequently observed in both RNA and DNA molecules. This motif is involved specifically in various biological functions (e.g., gene expression and regulation). To understand how these hairpin motifs perform their functions, it is important to study their structures. Compared to protein structural motifs, structures of nucleic acid hairpins are less known. Based on a set of reduced coordinates for describing nucleic acid structures and a sampling algorithm that equilibrates structures using Metropolis Monte Carlo simulation, we developed a method to model nucleic acid hairpin structures. This method was used to predict the structure of a DNA hairpin with a single-guanosine loop. The lowest energy structure from the ensemble of 200 sampled structures has a RMSD of <1.5 Å, from the structure determined using NMR. Additional constraints for the loop bases were introduced for modeling an RNA hairpin with two nucleotides in the loop. The modeled structure of this RNA hairpin has extensive base stacking and an extra hydrogen bond (between the CYT in the loop and a phosphate oxygen), as observed in the NMR structure. ImagesFIGURE 12

Tung, Chang-Shung

1997-01-01

262

Nucleic acid scavengers inhibit thrombosis without increasing bleeding  

PubMed Central

Development of effective, yet safe, antithrombotic agents has been challenging because such agents increase the propensity of patients to bleed. Recently, naturally occurring polyphosphates such as extracellular DNA, RNA, and inorganic polyphosphates have been shown to activate blood coagulation. In this report, we evaluate the anticoagulant and antithrombotic activity of nucleic acid-binding polymers in vitro and in vivo. Such polymers bind to DNA, RNA, and inorganic polyphosphate molecules with high affinity and inhibit RNA- and polyphosphate-induced clotting and the activation of the intrinsic pathway of coagulation in vitro. Moreover, [NH2(CH2)2NH2]?(G = 3);dendri PAMAM(NH2)32 (PAMAM G-3) prevents thrombosis following carotid artery injury and pulmonary thromboembolism in mice without significantly increasing blood loss from surgically challenged animals. These studies indicate that nucleic acid-binding polymers are able to scavenge effectively prothrombotic nucleic acids and other polyphosphates in vivo and represent a new and potentially safer class of antithrombotic agents.

Jain, Shashank; Pitoc, George A.; Holl, Eda K.; Zhang, Ying; Borst, Luke; Leong, Kam W.; Lee, Jaewoo; Sullenger, Bruce A.

2012-01-01

263

Nucleic acid sample preparation using spontaneous biphasic plug flow.  

PubMed

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 pipet that are laborious and time-consuming, making the procedure 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 polymerase chain reaction (PCR) amplification showed similar threshold cycle values as those obtained from a commercially available kit. Human immunodeficiency virus (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 was determined. 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

2013-09-17

264

Nucleic acid-induced antiviral immunity in shrimp.  

PubMed

Vertebrates detect viral infection predominantly by sensing viral nucleic acids to produce type I interferon (IFN). In invertebrates, it has been believed that the IFN system is absent and RNA interference is a sequence-specific antiviral pathway. In this study, we found that injection of nucleic acid mimics poly(I:C), poly(C:G), CL097, poly C and CpG-DNA, afforded shrimp antiviral immunity, which is similar to the vertebrate IFN system. Using suppression subtractive hybridization (SSH) method, 480 expression sequence tags were identified to be involved in the poly(I:C)-induced antiviral immunity of the model crustacean Litopenaeus vannamei, and 41% of them were new genes. In the SSH libraries, several IFN system-related genes such as dsRNA-dependent protein kinase PKR, Toll-like receptor 3 (TLR3) and IFN?-inducible protein 30 were identified. L. vannamei IKK?, whose vertebrate homologs are central regulators of the IFN-producing pathway, could significantly activate IFN reporter genes in HEK293T cells. In crustacean databases, many genes homologous to genes of the vertebrate IFN response, such as IRFs, PKR, ADAR (adenosine deaminase, RNA-specific) and other interferon-stimulated genes (ISGs) were discovered. These results suggest that shrimp may possess nucleic acid-induced antiviral immunity. PMID:23773856

Wang, Pei-Hui; Yang, Li-Shi; Gu, Zhi-Hua; Weng, Shao-Ping; Yu, Xiao-Qiang; He, Jian-Guo

2013-09-01

265

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.

Giatrellis, Sarantis; Nounesis, George

2011-01-01

266

Molecular assembly for high-performance bivalent nucleic acid inhibitor  

PubMed Central

It is theorized that multivalent interaction can result in better affinity and selectivity than monovalent interaction in the design of high-performance ligands. Accordingly, biomolecular engineers are increasingly taking advantage of multivalent interactions to fabricate novel molecular assemblies, resulting in new functions for ligands or enhanced performance of existing ligands. Substantial efforts have been expended in using small molecules or epitopes of antibodies for designing multifunctional or better-performing ligands. However, few attempts to use nucleic acid aptamers as functional domains have been reported. In this study, we explore the design of bivalent nucleic acid ligands by using thrombin and its aptamers as the model by which to evaluate its functions. By assembling two thrombin-binding aptamers with optimized design parameters, this assembly has resulted in the successful development of a nucleic acid-based high-performance bivalent protein inhibitor. Our experimentation proved (i) that the simultaneous binding of two aptamers after linkage achieved 16.6-fold better inhibition efficiency than binding of the monovalent ligand and (ii) that such an improvement originated from changes in the kinetics of the binding interactions, with a koff rate ?1/50 as fast. In addition, the newly generated aptamer assembly is an excellent anticoagulant reagent when tested with different samples. Because this optimized ligand design offers a simple and noninvasive means of accomplishing higher performance from known functional aptamers, it holds promise as a potent antithrombin agent in the treatment of various diseases related to abnormal thrombin activities.

Kim, Youngmi; Cao, Zehui; Tan, Weihong

2008-01-01

267

The role of immunostimulatory nucleic acids in septic shock  

PubMed Central

Sepsis and its associated syndromes represent the systemic host response to severe infection and is manifested by varying degrees of hypotension, coagulopathy, and multiorgan dysfunction. Despite great efforts being made to understand this condition and designing therapies to treat sepsis, mortality rates are still high in septic patients. Characterization of the complex molecular signaling networks between the various components of host-pathogen interactions, highlights the difficulty in identifying a single driving force responsible for sepsis. Although triggering the inflammatory response is generally considered as protective against pathogenic threats, the interplay between the signaling pathways that are induced or suppressed during sepsis may harm the host. Numerous surveillance mechanisms have evolved to discriminate self from foreign agents and accordingly provoke an effective cellular response to target the pathogens. Nucleic acids are not only an essential genetic component, but sensing their molecular signature is also an important quality control mechanism which has evolved to maintain the integrity of the human genome. Evidence that has accumulated recently indicated that distinct pattern recognition receptors sense nucleic acids released from infectious organisms or from damaged host cells, resulting in the modulation of intracellular signalling cascades. Immunoreceptor-mediated detection of these nucleic acids induces antigen-specific immunity, secretion of proinflammatory cytokines and reactive oxygen/nitrogen species and thus are implicated in a range of diseases including septic shock.

Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

2012-01-01

268

Differential fluorescence quenching of fluorescent nucleic acid base analogues by native nucleic acid monophosphates.  

PubMed

Fluorescent nucleic acid base analogues (FBAs) are used widely as probes of DNA and RNA structure and dynamics. Of increasing utility are the pteridone adenosine analogues (6MAP, DMAP) and pteridine guanosine analogues (3MI, 6MI). These FBAs (collectively referred to as PTERs) are useful, in part, because their fluorescence quantum yields, Phi(f), are modulated by base stacking with native bases (NBs), making them sensitive reporters of DNA structure. The quenching mechanism has been hypothesized to be photoinduced electron transfer following selective excitation of the FBA, but hard evidence for this has been lacking. The degree of quenching shows some dependence on the neighboring bases, but there has been no real determination as to whether FBA*:NB complexes satisfy the basic thermodynamic requirement for spontaneous PET: a negative free energy for the electron transfer reaction. Indeed, quenching may result from entirely different mechanisms. To address these questions, Stern-Volmer (S-V) experiments were performed using the native-base monophosphate nucleotides (NMPs) GMP, AMP, CMP, and dTMP in aqueous solutions as quenchers to obtain quenching rate constants, k(q). Cyclic voltammetry (CV) and optical absorption and emission data of the PTERS were obtained in aprotic organic solvents. These data were used to obtain excited-state redox potentials from which electron transfer free energies were derived using the Rehm-Weller equation. The reorganization energies for PET were obtained using the Scandola-Balzani equation, taking into account the free energy contribution due to water. 6MAP*, DMAP*, and 3MI* gave negative free energies between -0.1 and -0.2 eV and reorganization energies of about 0.13 eV. They all displayed ET activation energies below the accessible thermal energy (0.038 eV = 3/2k(B)T, where k(B) is Boltzmann's constant) for all NMPs with the exception of CMP, whose activation barrier was only about 35% higher (approximately 0.05 eV). Thus, we conclude that these PTERs act as electron acceptors and promote NMP oxidation. However, 6MI* had positive ET free energies for all NMPs with the exception of GMP (and then only for nucleobase oxidation). The magnitudes of these free energies (> or = 0.45 eV for AMP, CMP, and dTMP) suggest that 6MI* may not quenched by PET. PMID:20387838

Narayanan, Madhavan; Kodali, Goutham; Singh, Vijay; Xing, Yangjun; Hawkins, Mary E; Stanley, Robert J

2010-05-01

269

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

Microsoft Academic Search

The bulged insertions of (R )-1-O-(pyren-1-ylmethyl)- glycerol (monomer P) in two complementary 8mer DNA strands (intercalating nucleic acids) opposite to each other resulted in the formation of an easily denaturing duplex, which had lower thermal stability (21.0? C) than the wild-type double-stranded DNA (dsDNA, 26.0? C), but both modified oligodeoxy- nucleotides had increased binding affinity toward complementary single-stranded DNA (ssDNA)

Vyacheslav V. Filichev; Birte Vester; Lykke H. Hansen; Erik B. Pedersen

2005-01-01

270

Nucleic Acid Homology in the Genus Mycobacterium  

PubMed Central

Immobilized deoxyribonucleic acid (DNA) preparations from eight species of mycobacteria were reacted with labeled reference DNA from Mycobacterium tuberculosis and M. kansasii. All mycobacteria showed some degree of homology. Immobilized DNA from Pseudomonas multivorans, which has a guanine plus cytosine content within the range established for the mycobacteria, showed no significant binding with either reference system. Relative per cent binding and thermal stability of bound DNA were used as parameters for determining degrees of relatedness among members of the genus. When these relationships were compared with those established by numberical taxonomic methods, a high correlation was found.

Gross, Wendy M.; Wayne, Lawrence G.

1970-01-01

271

[Charge transfer complexes of nucleic acid purine bases with benzoquinone. Evidence that nucleic acid components are pi-donors].  

PubMed

The absorption bands of the charge transfer complexes between nucleic acid, purine components and some of their derivatives (9CH3--Ade, Ado, dAdo, pA, App, Guo, Gpp, Gppp, Cof, Hyp) as electron donors and p-benzoquinone as the acceptor were obtained by the differential method in water solution at 293 K. These bands lie in the range 330--430 nm and have all characteristic properties of the charge transfer bands. All points of the relationships between the energy corresponding to the charge transfer band maximum (hvct) and the donor ionization potential (Id) for the nucleic acid components and their derivatives fall on the same straight line for the pi-donors (the aromatic molecules), known from literature data, which differ markedly from the same straight line for n-donors (the alifatic amines), obtained in this work. It shows that the investigated nucleic acid components and their derivatives are the pi-donors in the charge transfer complexes with p-benzoquinone. PMID:7242539

Malevski?, A A; Rapoport, V L; Tret'iakov, A N

1981-01-01

272

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

PubMed

Two G-quadruplex forming sequences, 5'-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (twisted intercalating nucleic acid, TINA). Incorporation of LNA or INA/TINA monomers provide as much as 8-fold improvement of anti-HIV-1 activity. We demonstrate for the first time a detailed analysis of the effect the incorporation of INA/TINA monomers in quadruplex forming oligonucleotides (QFOs) and the effect of LNA monomers in the context of biologically active QFOs. In addition, recent literature reports and our own studies on the gel retardation of the phosphodiester analogue of T30177 led to the conclusion that this sequence forms a parallel, dimeric G-quadruplex. Introduction of the 5'-phosphate inhibits dimerisation of this G-quadruplex as a result of negative charge-charge repulsion. Contrary to that, we found that attachment of the 5'-O-DMT-group produced a more active 17-mer sequence that showed signs of aggregation-forming multimeric G-quadruplex species in solution. Many of the antiviral QFOs in the present study formed more thermally stable G-quadruplexes and also high-order G-quadruplex structures which might be responsible for the increased antiviral activity observed. PMID:21062811

Pedersen, Erik B; Nielsen, Jakob T; Nielsen, Claus; Filichev, Vyacheslav V

2011-03-01

273

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.

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

2011-01-01

274

Nucleic acid encoding MIP-1.alpha. Lymphokine  

US Patent & Trademark Office Database

Full length cDNAs, L2G25B and 4-1BB, were isolated and sequenced. The cDNA L2G25B encodes for the lymphokine, macrophage inflammatory protein-1.alpha. or MIP-1.alpha.. The studies disclosed herein suggest that MIP-1.alpha. and MIP-.beta. can, through rapid action, modulate early myeloid progenitor cell proliferation. Recombinant proteins have been produced for the cytokine, L2G25BP (Macrophage Inflammatory Protein-1.alpha., MIP-1.alpha.). By employing the recombinant protein (rMIP-1.alpha.), receptors for MIP-1.alpha. were identified on Con A-stimulated and unstimulated CTLL-R8, a T-cell line, and LPS-stimulated RAW 264.7, a macrophage-cell line. Purified recombinant murine macrophage inflammatory protein-1 alpha (rmuMIP-.alpha.), was assessed for effects on proliferation of granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells. The results suggest that rmuMIP-1.alpha. has myelosuppressive activity in vivo. The cDNA clone, called 4-1BB, is an inducible receptor-like sequence found in both cytolytic and helper T-cells.

2002-03-12

275

The peptide nucleic acids as probes for chromosomal analysis: application to human oocytes, polar bodies and preimplantation embryos  

Microsoft Academic Search

Peptide nucleic acids (PNA) are synthetic DNA mimics based on an uncharged polyamide backbone, which hybridize with complementary DNA with high affinity and specificity. PNA have recently become recognized as efficient tools for in situ chromosomal identification. In the present study, this new approach has been tried on isolated human oocytes, polar bodies and blastomeres. Using centromeric PNA probes specific

P. Paulasova; B. Andreo; J. Diblik; M. Macek; F. Pellestor

2004-01-01

276

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

277

Origin of Overstretching Transitions in Single-Stranded Nucleic Acids  

NASA Astrophysics Data System (ADS)

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

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

2013-11-01

278

Origin of overstretching transitions in single-stranded nucleic acids.  

PubMed

We combined single-molecule force spectroscopy with nuclear magnetic resonance measurements and molecular mechanics simulations to examine overstretching transitions in single-stranded nucleic acids. In single-stranded DNA and single-stranded RNA there is a low-force transition that involves unwinding of the helical structure, along with base unstacking. We determined that the high-force transition that occurs in polydeoxyadenylic acid single-stranded DNA is caused by the cooperative forced flipping of the dihedral angle formed between four atoms, O5'-C5'-C4'-C3' (? torsion), in the nucleic acid backbone within the canonical B-type helix. The ? torsion also flips under force in A-type helices, where the helix is shorter and wider as compared to the B-type helix, but this transition is less cooperative than in the B type and does not generate a high-force plateau in the force spectrums of A-type helices. We find that a similar high-force transition can be induced in polyadenylic acid single-stranded RNA by urea, presumably due to disrupting the intramolecular hydrogen bonding in the backbone. We hypothesize that a pronounced high-force transition observed for B-type helices of double stranded DNA also involves a cooperative flip of the ? torsion. These observations suggest new fundamental relationships between the canonical structures of single-and double-stranded DNA and the mechanism of their molecular elasticity. PMID:24237568

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

2013-11-01

279

Extracellular Nucleic Acids of the Marine Phototrophic Bacterium Rhodovulum sulfidophilum and Related Bacteria: Physiology and Biotechnology  

Microsoft Academic Search

\\u000a Extracellular nucleic acids of high molecular weight are present ubiquitously throughout the environment, such as seawater\\u000a and soil. These nucleic acids were formerly thought to be derived from cells by cell death, but recent studies have shown\\u000a that they are at least partly derived from the active release of nucleic acids from some bacterial cells. Marine phototrophic\\u000a bacteria, Rhodovulum sulfidophilum

Yo Kikuchi

280

Use of carbonyl group addition--elimination reactions for synthesis of nucleic acid conjugates.  

PubMed

This review outlines the synthesis of covalent conjugates of oligonucleotides and their analogues that are obtained by reactions of carbonyl compounds with various nucleophiles such as primary amines, N-alkoxyamines, hydrazines, and hydrazides. The products linked by imino, oxime, hydrazone, or thiazolidine groups are shown to be useful intermediates for a wide range of chemical biology applications. Methods for their preparation, isolation, purification, and analysis are highlighted, and the comparative stabilities of the respective linkages are evaluated. The relative merits of incorporation of a carbonyl group, particularly an aldehyde group, into either the oligonucleotide or the ligand parts are considered. Examples of harnessing of aldehyde-nucleophile coupling for the labeling of nucleic acids are given, as well as their conjugation to various biomolecules (e.g. peptides and small molecule ligands), site-specific cross-linking of oligonucleotides to nucleic acid-binding proteins, assembly of multibranched supramolecular structures, and immobilization on functionalized surfaces. Future perspectives of bioconjugation and complex molecular engineering via carbonyl group addition-elimination reactions in nucleic acids chemistry are discussed. PMID:15898711

Zatsepin, Timofei S; Stetsenko, Dmitry A; Gait, Michael J; Oretskaya, Tatiana S

2005-01-01

281

Development and performance evaluation of calf diarrhea pathogen nucleic acid purification and detection workflow.  

PubMed

Calf diarrhea (scours) is a primary cause of illness and death in young calves. Significant economic losses associated with this disease include morbidity, mortality, and direct cost of treatment. Multiple pathogens are responsible for infectious diarrhea, including, but not limited to, Bovine coronavirus (BCV), bovine Rotavirus A (BRV), and Cryptosporidium spp. Identification and isolation of carrier calves are essential for disease management. Texas Veterinary Medical Diagnostic Laboratory current methods for calf diarrhea pathogen identification include electron microscopy (EM) for BCV and BRV and a direct fluorescent antibody test (DFAT) for organism detection of Cryptosporidium spp. A workflow was developed consisting of an optimized fecal nucleic acid purification and multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) for single tube concurrent detection of BCV, BRV, and Cryptosporidium spp., and an internal control to monitor nucleic acid purification efficacy and PCR reagent functionality. In "spike-in" experiments using serial dilutions of each pathogen, the analytical sensitivity was determined to be <10 TCID(50)/ml for BCV and BRV, and <20 oocysts for Cryptosporidium spp. Analytical specificity was confirmed using Canine and Feline coronavirus, Giardia spp., and noninfected bovine purified nucleic acid. Diagnostic sensitivity was ?98% for all pathogens when compared with respective traditional methods. The results demonstrate that the newly developed assay can purify and subsequently detect BCV, BRV, and Cryptosporidium spp. concurrently in a single PCR, enabling simplified and streamlined calf diarrhea pathogen identification. PMID:22914823

Schroeder, Megan E; Bounpheng, Mangkey A; Rodgers, Sandy; Baker, Rocky J; Black, Wendy; Naikare, Hemant; Velayudhan, Binu; Sneed, Loyd; Szonyi, Barbara; Clavijo, Alfonso

2012-09-01

282

Ion-Mediated Nucleic Acid Helix-Helix Interactions  

PubMed Central

Salt ions are essential for the folding of nucleic acids. We use the tightly bound ion (TBI) model, which can account for the correlations and fluctuations for the ions bound to the nucleic acids, to investigate the electrostatic free-energy landscape for two parallel nucleic acid helices in the solution of added salt. The theory is based on realistic atomic structures of the helices. In monovalent salt, the helices are predicted to repel each other. For divalent salt, while the mean-field Poisson-Boltzmann theory predicts only the repulsion, the TBI theory predicts an effective attraction between the helices. The helices are predicted to be stabilized at an interhelix distance ?26–36 Å, and the strength of the attractive force can reach ?0.37 kBT/bp for helix length in the range of 9–12 bp. Both the stable helix-helix distance and the strength of the attraction are strongly dependent on the salt concentration and ion size. With the increase of the salt concentration, the helix-helix attraction becomes stronger and the most stable helix-helix separation distance becomes smaller. For divalent ions, at very high ion concentration, further addition of ions leads to the weakening of the attraction. Smaller ion size causes stronger helix-helix attraction and stabilizes the helices at a shorter distance. In addition, the TBI model shows that a decrease in the solvent dielectric constant would enhance the ion-mediated attraction. The theoretical findings from the TBI theory agree with the experimental measurements on the osmotic pressure of DNA array as well as the results from the computer simulations.

Tan, Zhi-Jie; Chen, Shi-Jie

2006-01-01

283

Cationic lipid nanosystems as carriers for nucleic acids.  

PubMed

Solid lipid nanoparticles (SLNs) consisting of tristearin or tribehenin, and monoolein aqueous dispersions (MADs) consisting of glyceryl-monoolein have been studied as potential nanocarriers for nucleic acids. The cationic character of nanocarriers was obtained by adding cationic surfactants, such as diisobutylphenoxyethyl-dimethylbenzyl ammonium chloride (DEBDA) or PEG-15 Cocopolyamine (PCPA), to the lipid composition. The products were characterised in terms of size and morphology by Cryo-TEM and PCS. The charge properties were determined by measuring the zeta potential. Our experimental protocol enabled us to obtain homogeneous and stable cationic nanosystems within 3-6 months of production. Assessment of cytotoxicity on HepG2 cells by MTT assays indicated that MAD preparations were less toxic than SLN, and in general PCPA-containing formulations are less cytotoxic than DEBDA-containing ones. The formation of electrostatic complexes with salmon sperm or plasmid DNA, used as model nucleic acids, was evaluated by electrophoresis on agarose gel. The results confirmed that all the formulations studied are able to form the complex. Finally, we investigated the ability of SLN and MAD to deliver DNA into HepG2 cells, and to this purpose we exploited expression plasmids for green fluorescent protein or firefly luciferase. Although with reduced efficiency, the results showed that the produced nanocarriers are able to convey plasmids into cells. The data obtained encourage further study aimed at improving these new formulations and proposing them as novel in vitro transfection reagents with potential application to in vivo delivery of nucleic acids. PMID:24120492

Cortesi, Rita; Campioni, Matteo; Ravani, Laura; Drechsler, Markus; Pinotti, Mirko; Esposito, Elisabetta

2014-01-25

284

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

PubMed

Chemical conjugations of nucleic acids with macromolecules or small molecules are common approaches to study nucleic acids in chemistry and biology and to exploit nucleic acids for medical applications. The conjugation of nucleic acids such as oligonucleotides with peptides is especially useful to circumvent cell delivery and specificity problems of oligonucleotides as therapeutic agents. However, current approaches are limited and inefficient in their ability to afford peptide-oligonucleotide conjugates (POCs). Here, we report an effective and reproducible approach to prepare POCs and other nucleic acid conjugates based on a newly developed nucleic acid phosphoramidation method. The development of a new nucleic acid phosphoramidation reaction was achieved by our successful synthesis of a novel amine-containing biotin derivative used to systematically optimize the reactions. The improved phosphoramidation reactions dramatically increased yields of nucleic acid-biotin conjugates up to 80% after 3 h reaction. Any nucleic acids with a terminal phosphate group are suitable reactants in phosphoramidation reactions to conjugate with amine-containing molecules such as biotin and fluorescein derivatives, proteins, and, most importantly, peptides to enable the synthesis of POCs for therapeutic applications. Polymerase chain reactions (PCRs) to study incorporation of biotin or fluorescein-tagged DNA primers into the reaction products demonstrated that appropriate controls of nucleic acid phosphoramidation reactions incur minimum adverse effects on inherited base-pairing characteristics of nucleotides in nucleic acids. The phosphoramidation approach preserves the integrity of hybridization specificity in nucleic acids when preparing POCs. By retaining integrity of the nucleic acids, their effectiveness as therapeutic reagents for gene silencing, gene therapy, and RNA interference is ensured. The potential for POC use was demonstrated by two-step phosphoramidation reactions to successfully synthesize nucleic acid-tetraglycine conjugates. In addition, phosphoramidation reactions provided a facile approach to prepare nucleic acid-BSA conjugates with good yields. In summary, the new approach to phosphoramidation reactions offers a universal method to prepare POCs and other nucleic acid conjugates with high yields in aqueous solutions. The methods can be easily adapted to typical chemistry or biology laboratory setups which will expedite the applications of POCs for basic research and medicine. PMID:20690641

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

2010-09-15

285

Methods, systems and kits for detecting protein-nucleic acid interactions  

US Patent & Trademark Office Database

Methods, systems and kits for detecting protein-nucleic acid interactions, in particular, detecting the genomic location to near-base pair resolution at which a particular protein (e.g., transcription factor) binds includes combining steps of a conventional chromatin immunoprecipitation (ChIP) assay with use of an exonuclease that digests nucleic acid strands in the 5'-3' or 3'-5' direction until it reaches a bound protein including a protein crosslinked to the nucleic acid. Proteins that inefficiently crosslink to a nucleic acid and thus are very difficult to detect, are expected to be significantly detected by the kits and methods described herein.

2013-02-05

286

Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids  

Microsoft Academic Search

An analogous series of cationic lipids (1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA) and 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA)) possessing 0, 1, 2 or 3 double bonds per alkyl chain respectively, was synthesized to determine the correlation between lipid saturation, fusogenicity and efficiency of intracellular nucleic acid delivery. 31P-NMR analysis suggests that as saturation increases, from 2 to 0 double bonds, lamellar (L?) to

James Heyes; Lorne Palmer; Kaz Bremner; Ian MacLachlan

2005-01-01

287

Antibody-linked Spherical Nucleic Acids for Cellular Targeting  

PubMed Central

Spherical nucleic acid (SNAs) constructs are promising new single entity gene regulation materials capable of both cellular transfection and gene knockdown, but thus far are promiscuous structures, exhibiting excellent genetic but little cellular selectivity. In this communication, we describe a strategy to impart targeting capabilities to these constructs through non-covalent functionalization with a complementary antibody-DNA conjugate. As a proof-of concept, we designed HER2-targeting SNAs and demonstrated that such structures exhibit cell type selectivity in terms of their uptake, and significantly greater gene knockdown in cells overexpressing the target antigen as compared to the analogous antibody-free and off-target materials.

Zhang, Ke; Hao, Liangliang; Hurst, Sarah J.; Mirkin, Chad A.

2012-01-01

288

Interaction of bleomycin and its oligonucleotide derivatives with nucleic acids  

NASA Astrophysics Data System (ADS)

Various aspects of interaction of the antitumour glycopeptide antibiotic bleomycin with nucleic acids are considered. Data on equilibrium binding parameters obtained by various physicochemical methods have been collected and compared. The contribution of N- and C-terminal domains of the glycopeptide molecule to the binding with DNA and sequence specificity of DNA cleavage are discussed. Data on a recently created new class of compounds — bleomycin — oligonucleotide conjugates — are presented. These compounds, like antibiotics, possess DNA-cleaving activity (also in a catalytic manner) together with high selectivity towards a selected nucleotide sequence. The bibliography includes 267 references.

Sergeyev, D. S.; Zarytova, V. F.

1996-04-01

289

Ion trap collision-induced dissociation of locked nucleic acids  

Microsoft Academic Search

Gas-phase dissociation of model locked nucleic acid (LNA) oligonucleotides and functional LNA-DNA chimeras have been investigated\\u000a as a function of precursor ion charge state using ion trap collision-induced dissociation (CID). For the model LNA 5 and 8\\u000a mer, containing all four LNA monomers in the sequence, cleavage of all backbone bonds, generating a\\/w-, b\\/x-, c\\/y-, and d\\/z-ions,\\u000a was observed with

Teng-yi Huang; Anastasia Kharlamova; Scott A. McLuckey

2010-01-01

290

Conducting Polymer Based Nucleic Acid Sensor for Environment Monitoring  

NASA Astrophysics Data System (ADS)

Nucleic acid sensor based on polyaniline has been fabricated by covalently immobilizing double stranded calf thymus (dsCT) DNA onto perchlorate (ClO-4) doped polyaniline (PANI) film deposited onto indium-tin-oxide (ITO) glass plate using 1-(3-(dimethylamino) propyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxyl succinimide (NHS) chemistry. These dsCT-DNA-PANI/ITO and PANI/ITO electrodes have been characterized using square wave voltammetry, electrochemical impedance, and Fourier-transform-infra-red (FTIR) measurements. This disposable dsCT-DNA-PANI/ITO bioelectrode is stable for about four months, can be used to detect arsenic trioxide (0.1ppm) in 30s.

Malhotra, Bansi Dhar; Prabhakar, Nirmal; Solanki, Pratima R.

291

Integrating nano-optical biosensors into nucleic acid testing devices  

NASA Astrophysics Data System (ADS)

Nano optical biosensors employ the interaction between biomolecules and light confined in nanometer scale structures to report the bio-recognition events. This small scale sensing area/volume can ensure that small amount of biorecognition events could be detected. The exceptional sensitivity and high spatial density of nano optical biosensors make them unique in practical applications in nucleic acid detection. Lab-on-a-Chip systems provide the capabilities of separation, cell lysing, polymerase chain reaction (PCR), allowing finishing bio agent detection processes on a chip. In this paper, we present our recent efforts on integrating some novel nanooptical biosensors into Lab-on-a-Chip systems and some preliminary test results.

Wei, Qi-Huo; Gu, Jian; Chou, Chia-Fu; Zenhausern, Frederic

2004-06-01

292

BGL7 beta-glucosidase and nucleic acids encoding the same  

DOEpatents

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

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

2008-08-05

293

BGL4 beta-glucosidase and nucleic acids encoding the same  

DOEpatents

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

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

2011-12-06

294

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

DOEpatents

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

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

2008-03-18

295

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

DOEpatents

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

Dunn-Coleman, Nigel; Ward, Michael

2012-10-02

296

BGL7 beta-glucosidase and nucleic acids encoding the same  

DOEpatents

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

Dunn-Coleman, Nigel; Ward, Michael

2013-01-29

297

BGL3 beta-glucosidase and nucleic acids encoding the same  

DOEpatents

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

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

2011-06-14

298

BGL3 beta-glucosidase and nucleic acids encoding the same  

DOEpatents

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

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

2008-04-01

299

BGL3 beta-glucosidase and nucleic acids encoding the same  

DOEpatents

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

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

2007-09-25

300

BGL3 beta-glucosidase and nucleic acids encoding the same  

DOEpatents

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

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

2012-10-30

301

BGL6 beta-glucosidase and nucleic acids encoding the same  

DOEpatents

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

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

2009-09-01

302

Analysis of bases of rat-liver nucleic acids after administration of the carcinogen dimethylnitrosamine  

PubMed Central

Dimethylnitrosamine is metabolized to form an alkylating intermediate, which may have significance for its carcinogenic action. However, certain other compounds that are known to be highly mutagenic, including nitrous acid and hydroxylamine, might also be formed. Owing to the general reactivity of these compounds, it would be difficult to detect their formation in the intact animal. Instead, the nucleic acids of carcinogen-treated animals were examined for products of reaction with nitrous acid and hydroxylamine, i.e. for deamination of adenine and guanine, and formation of N6-hydroxycytosine, respectively. A double-labelling technique was used to detect very small amounts of the abnormal bases. The purine moieties of DNA in adult rat liver were labelled either with 14C or with 3H, by treating the neonatal animals with [14C]formate or with [3H]formate, and then allowing a period for normal growth. During this time, in liver, the labels were largely lost by metabolic turnover from cell components other than DNA. The pyrimidine moieties in DNA were labelled by treating the neonatal animals with [14C]orotate. The purine constituents of RNA of adult rat liver were labelled by repeated administration of [14C]- or [3H]-formate to the adult rats. The [14C]nucleic acid-labelled rat could then be treated with the carcinogen, and the [3H]nucleic acid-labelled animal could be used as a control. By this means the experimental and control tissues could be homogenized together in a single preparation, and the nucleic acids from the two tissues could be isolated, hydrolysed and analysed in a single sample. It was therefore possible to have an internal control for artifacts due to changes taking place in the nucleic acid bases during the experimental procedures. With this technique, the formation in vivo of 7-methylguanine in rat-liver DNA and RNA after administration of dimethylnitrosamine was confirmed, and no evidence was found for the formation of xanthine, hypoxanthine, N6-hydroxycytosine, or any other abnormal base.

Craddock, Valda M.; Magee, P. N.

1966-01-01

303

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

304

Analyzing and Building Nucleic Acid Structures with 3DNA  

PubMed Central

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at http://w3dna.rutgers.edu, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

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

2013-01-01

305

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.

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

2012-01-01

306

Mechanism for the endocytosis of spherical nucleic acid nanoparticle conjugates  

PubMed Central

Intracellular delivery of nucleic acids as gene regulation agents typically requires the use of cationic carriers or viral vectors, yet issues related to cellular toxicity or immune responses hamper their attractiveness as therapeutic candidates. The discovery that spherical nucleic acids (SNAs), polyanionic structures comprised of densely packed, highly oriented oligonucleotides covalently attached to the surface of nanoparticles, can effectively enter more than 50 different cell types presents a potential strategy for overcoming the limitations of conventional transfection agents. Unfortunately, little is known about the mechanism of endocytosis of SNAs, including the pathway of entry and specific proteins involved. Here, we demonstrate that the rapid cellular uptake kinetics and intracellular transport of SNAs stem from the arrangement of oligonucleotides into a 3D architecture, which supports their targeting of class A scavenger receptors and endocytosis via a lipid-raft–dependent, caveolae-mediated pathway. These results reinforce the notion that SNAs can serve as therapeutic payloads and targeting structures to engage biological pathways not readily accessible with linear oligonucleotides.

Choi, Chung Hang J.; Hao, Liangliang; Narayan, Suguna P.; Auyeung, Evelyn; Mirkin, Chad A.

2013-01-01

307

Prebiotically plausible mechanisms increase compositional diversity of nucleic acid sequences.  

PubMed

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

2012-05-01

308

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

309

Thermodynamics of RNA duplexes modified with unlocked nucleic acid nucleotides  

PubMed Central

Thermodynamics provides insights into the influence of modified nucleotide residues on stability of nucleic acids and is crucial for designing duplexes with given properties. In this article, we introduce detailed thermodynamic analysis of RNA duplexes modified with unlocked nucleic acid (UNA) nucleotide residues. We investigate UNA single substitutions as well as model mismatch and dangling end effects. UNA residues placed in a central position makes RNA duplex structure less favourable by 4.0–6.6?kcal/mol. Slight destabilization, by ?0.5–1.5?kcal/mol, is observed for 5?- or 3?-terminal UNA residues. Furthermore, thermodynamic effects caused by UNA residues are extremely additive with ?G°37 conformity up to 98%. Direct mismatches involving UNA residues decrease the thermodynamic stability less than unmodified mismatches in RNA duplexes. Additionally, the presence of UNA residues adjacent to unpaired RNA residues reduces mismatch discrimination. Thermodynamic analysis of UNA 5?- and 3?-dangling ends revealed that stacking interactions of UNA residues are always less favourable than that of RNA residues. Finally, circular dichroism spectra imply no changes in overall A-form structure of UNA–RNA/RNA duplexes relative to the unmodified RNA duplexes.

Pasternak, Anna; Wengel, Jesper

2010-01-01

310

Molecular polarization potential maps of the nucleic acid bases  

SciTech Connect

Ab initio calculations at the SCF level were carried out to compute the polarization potential map NM of the nucleic acid bases: cytosine, thymine, uracil, adedine, and guanine. For this purpose, the Dunning`s 9s5p basis set contracted to a split-valence, was selected to perform the calculations. The molecular polarization potential (MPP) at each point was evaluated by the difference between the interaction energy of the molecule with a unit point charge and the molecular electrostatic potential (MEP) at that point. MEPS and MPPS for the different molecules were computed with a density of 5 points/{Angstrom}{sup 2} on the van der Waals surface of each molecule, defined using the van der Waals radii. Due to the symmetry of the molecules, only half the points were computed. The total number of points calculated was 558 for cytosine, 621 for thymine, 526 for uracil, 666 for adenine, and 699 for guanine. The results of these calculations are analyzed in terms of their implications on the molecular interactions between pairs of nucleic acid bases. 23 refs., 5 figs., 1 tab.

Alkorta, I. [Instituto de Quimica Medica, Madrid (Spain)] [Instituto de Quimica Medica, Madrid (Spain); Perez, J.J. [ETS d`Enginyers Industrials, Barcelona (Spain)] [ETS d`Enginyers Industrials, Barcelona (Spain)

1996-01-05

311

Analyzing and building nucleic acid structures with 3DNA.  

PubMed

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at http://w3dna.rutgers.edu, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations. PMID:23644419

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

2013-01-01

312

Mechanism for the endocytosis of spherical nucleic acid nanoparticle conjugates.  

PubMed

Intracellular delivery of nucleic acids as gene regulation agents typically requires the use of cationic carriers or viral vectors, yet issues related to cellular toxicity or immune responses hamper their attractiveness as therapeutic candidates. The discovery that spherical nucleic acids (SNAs), polyanionic structures comprised of densely packed, highly oriented oligonucleotides covalently attached to the surface of nanoparticles, can effectively enter more than 50 different cell types presents a potential strategy for overcoming the limitations of conventional transfection agents. Unfortunately, little is known about the mechanism of endocytosis of SNAs, including the pathway of entry and specific proteins involved. Here, we demonstrate that the rapid cellular uptake kinetics and intracellular transport of SNAs stem from the arrangement of oligonucleotides into a 3D architecture, which supports their targeting of class A scavenger receptors and endocytosis via a lipid-raft-dependent, caveolae-mediated pathway. These results reinforce the notion that SNAs can serve as therapeutic payloads and targeting structures to engage biological pathways not readily accessible with linear oligonucleotides. PMID:23613589

Choi, Chung Hang J; Hao, Liangliang; Narayan, Suguna P; Auyeung, Evelyn; Mirkin, Chad A

2013-05-01

313

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

314

Targeting nucleic acid secondary structures by antisense oligonucleotides designed through in vitro selection.  

PubMed Central

Using an in vitro selection approach, we have isolated oligonucleotides that can bind to a DNA hairpin structure. Complex formation of these oligonucleotides with the target hairpin involves some type of triple-stranded structure with noncanonical interaction, as indicated by bandshift assays and footprinting studies. The selected oligomers can block restriction endonuclease cleavage of the target hairpin in a sequence-specific manner. We demonstrate that in vitro selection can extend the antisense approach to functional targeting of secondary structure motifs. This could provide a basis for interfering with regulatory processes mediated by a variety of nucleic acid structures. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

Mishra, R K; Le Tinevez, R; Toulme, J J

1996-01-01

315

Synthesis and hybridization properties of polyamide based nucleic acid analogues incorporating pyrrolidine-derived nucleoamino acids.  

PubMed

Ndelta-Fmoc protected nucleoamino acids of type I (Base = T, C, A) have been synthesized and employed as building blocks for the construction of novel polyamide based nucleic acid analogues. Homopyrimidine oligomer A binds to complementary RNA with significant affinity and in a sequence-specific fashion, while no binding was observed to complementary DNA. PMID:10853662

Altmann, K H; Hüsken, D; Cuenoud, B; García-Echeverría, C

2000-05-01

316

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.

Kurz, Matthias

2008-01-01

317

Solid-phase synthesis of peptide nucleic acids.  

PubMed

Peptide nucleic acids (PNA) were synthesized by a modified Merrifield method using several improvements. Activation by O-[benzotriazol-1-yl]-1,1,3,3-tetramethyluronium hexafluorophosphate in combination with in situ neutralization of the resin allowed efficient coupling of all four Boc-protected PNA monomers within 30 min. HPLC analysis of the crude product obtained from a fully automated synthesis of the model PNA oligomer H-CGGACTAAGTCCATTGC-Gly-NH2, indicated an average yield per synthetic cycle of 97.1%. N1-benzyloxycarbonyl-N6(3)-methylimidazole triflate substantially outperformed acetic anhydride as a capping reagent. The resin-bound PNAs were successfully cleaved by the 'low-high' trifluoromethanesulphonic acid procedure. PMID:9222994

Christensen, L; Fitzpatrick, R; Gildea, B; Petersen, K H; Hansen, H F; Koch, T; Egholm, M; Buchardt, O; Nielsen, P E; Coull, J; Berg, R H

1995-01-01

318

LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition  

Microsoft Academic Search

LNA (Locked Nucleic Acids), consisting of 2?-O,4?-C-methylene bicyclonucleoside monomers, is efficiently synthesized and its nucleic acid recognition potential evaluated for six different nucleobases, namely adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil. Unprecedented increases (+3 to +8 °C per modification) in the thermal stability of duplexes towards both DNA and RNA were obtained when evaluating mixed sequences of partly or fully

Alexei A. Koshkin; Sanjay K. Singh; Poul Nielsen; Vivek K. Rajwanshi; Ravindra Kumar; Michael Meldgaard; Carl Erik Olsen; Jesper Wengel

1998-01-01

319

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

320

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

Microsoft Academic Search

The current issue of Nucleic Acids Research includes descriptions of 179 databases, of which 95 are new. These databases (along with several molecular biology databases described in other journals) have been included in the Nucleic Acids Research online Molecular Biology Database Collection, bringing the total number of databases in the collection to 1170. In this introductory comment, we briefly describe

M. Y. Galperin; Guy Cochrane

2009-01-01

321

Instrument-free nucleic acid amplification assays for global health settings  

Microsoft Academic Search

Many infectious diseases that affect global health are most accurately diagnosed through nucleic acid amplification and detection. However, existing nucleic acid amplification tests are too expensive and complex for most low-resource settings. The small numbers of centralized laboratories that exist in developing countries tend to be in urban areas and primarily cater to the affluent. In contrast, rural area health

Paul Labarre; David Boyle; Kenneth Hawkins; Bernhard Weigl

2011-01-01

322

Neutral additives enhance the metal-chelate affinity adsorption of nucleic acids: Role of water activity  

Microsoft Academic Search

Immobilized metal-chelate affinity chromatography has been widely used in the purification of proteins, and we have recently found that it can also be applied to purification of nucleic acids through interactions involving exposed bases, especially purines. Here we report that the inclusion of moderate quantities of neutral solutes in the buffer substantially enhances the binding affinity of nucleic acids for

Ajish S. R. Potty; Joseph Y. Fu; Sindhu Balan; Barry L. Haymore; David J. Hill; George E. Fox; Richard C. Willson

2006-01-01

323

Dinuclear Cooper-Based Compounds and Ligamd for Nucleic Acid Scission and Anticancer Treatment.  

National Technical Information Service (NTIS)

The present invention is related to a novel method for splitting nucleic acids at specific points on a complementary nucleic acid segment using a dinuclear copper-based compound of Formula I. Additionally, the present invention is related to a novel treat...

S. E. Rokita K. D. Karlin K. J. Humphreys L. Li N. N. Murthy

2002-01-01

324

Final Report Nucleic Acid System: Hybird PCR and Multiplex Assay Project Phase II.  

National Technical Information Service (NTIS)

This report covers phase 2 (year 2) of the Nucleic Acid System - Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition...

R. P. Koopman R. G. Langlois S. Nasarabadi W. J. Benett B. W. Colston D. C. Johnson S. B. Brown P. L. Stratton F. P. Milanovich

2002-01-01

325

Fluorometric quantification of RNA and DNA in solutions containing both nucleic acids  

Microsoft Academic Search

A fluorescence-based method for quantitative determination of RNA and DNA in probes containing both nucleic acids has been developed. The total concentration of nucleic acids is determined using SYBR Green II dye under conditions providing independent binding of the fluorophore with DNA and RNA. The concentration of DNA is specifically measured using the Hoechst 33258 dye and the RNA concentration

Evgeniy S Morozkin; Pavel P Laktionov; Elena Y Rykova; Valentin V Vlassov

2003-01-01

326

[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

327

Artificial Functional Nucleic Acids: Aptamers, Ribozymes, and Deoxyribozymes Identified by In Vitro Selection  

NASA Astrophysics Data System (ADS)

The discovery of natural RNA catalysts (ribozymes) inspired the use of in vitro selection methodology to develop artificial functional nucleic acids (FNAs). In vitro selection is the experimental process by which large random-sequence pools of RNA or DNA are used as the starting point to identify particular nucleic acid sequences that have desired functions. When this function is binding of a molecular target, the functional nucleic acid is an RNA or DNA “aptamer.” When this function is catalysis of a chemical reaction, the functional nucleic acid is a “ribozyme” or “deoxyribozyme”; these are collectively termed “nucleic acid enzymes.” Since the first in vitro selection experiments in 1990, a wide variety of aptamers and nucleic acid enzymes have been identified. This chapter describes how aptamers, ribozymes, and deoxyribozymes are obtained by in vitro selection methodologies. Also addressed are the scope of the molecular targets that are bound and the chemical reactions that are catalyzed. Biochemical and structural characterizations of aptamers and nucleic acid enzymes are discussed. A final section introduces aptazymes, which are allosterically regulated nucleic acid enzymes.

Silverman, Scott K.

328

Label-free detection of nucleic acid and protein microarrays by scanning Kelvin nanoprobe  

Microsoft Academic Search

A high-resolution scanning Kelvin nanoprobe is introduced as an alternative technique to the conventional fluorescence and mass spectrometric detection methods currently employed in nucleic acid and protein microarray technology. The new instrument is capable of the highly sensitive discernment of surface biochemical events taking place at molecular level such as nucleic acid hybridization and antibody–antigen interaction. The method involves measurement

Michael Thompson; Larisa-Emilia Cheran; Mingquan Zhang; Melissa Chacko; Hong Huo; Saman Sadeghi

2005-01-01

329

Circulating tumour-derived nucleic acids in cancer patients: potential applications as tumour markers  

PubMed Central

Tumour-associated changes have been observed in the circulating nucleic acids of cancer patients and have been proposed to be useful for the detection and monitoring of cancers. In this review, different approaches for detecting tumour-associated nucleic acids in the circulation and their potential applications as tumour markers are discussed.

Chan, K C A; Lo, Y M D

2007-01-01

330

Use of Nucleic-Acid Homologies in the Taxonomy of Anaerobic Bacteria  

Microsoft Academic Search

Nucleic acid homology studies are providing a common base for establishing bacterial groups. Few phenotypic characteristics have consistently correlated with homology data among the various groups of organisms that we have investigated. However, there are correlations that are specific for a given group of bacteria such that nucleic-acid homology data can be used to select those phenotypic properties that will

JOHN L. JOHNSON

1973-01-01

331

Studies on nucleic acid metachromasy. II. Metachromatic and orthochromatic staining by toluidine blue of nucleic acids in tissue sections.  

PubMed

Acrolein-fixed, polyester wax-embedded tissue sections showed excellent preservation of light microscopic architecture and, when stained with toluidine blue, intense color contrast between DNA, which stained orthochromatically, and RNA, which stained metachromatically. This method has practical value for differentiating DNA from RNA in the same section. The color contrast was impaired by substituting formaldehyde for acrolein or paraffin for polyester wax, and was negligible in tissues fixed in formaldehyde or Carnoy's fluid and embedded in paraffin. Quality of structural preservation paralleled degree of color contrast. Metachromatic staining can be analysed, by the quantitative parameters of Bradley and colleagues, to provide inferences regarding the conformation of biopolymers in tissue sections. Comparison of the nucleic acid color contrasts in toluidine blue-stained sections with titrations of fixative-treated nucleic acids against toluidine blue in solution indicated a greater difference in conformation between DNA- and RNA-protein in acrolein-polyester sections than between acrolein-treated free DNA and RNA in solution. This is supported by recent evidence that the conformation of ribosomal RNA is quite different in whole ribosomes from that assumed by the same RNA free in solution. The acrolein-polyester method may enhance color contrast by providing superior preservation of ordered nucleoprotein conformations. PMID:4160917

Feder, N; Wolf, M K

1965-11-01

332

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

333

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

PubMed

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

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

2014-06-01

334

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

PubMed Central

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

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

2012-01-01

335

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

PubMed Central

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

Marcia, Marco; Humphris-Narayanan, Elisabeth; Keating, Kevin S.; Somarowthu, Srinivas; Rajashankar, Kanagalaghatta; Pyle, Anna Marie

2013-01-01

336

Synthesis of pyrrolidine-based oxy-peptide nucleic acids that contain four bases and their properties.  

PubMed

Adenine, guanine, thymine and cytosine monomers of oxy-peptide nucleic acid that contain a pyrrolidine ring in the main chain have been synthesized for use in the synthesis of novel peptide nucleic acid. PMID:17150561

Kitamatsu, Mizuki; Shigeyasu, Masanori; Okada, Tomoyuki; Saitoh, Mamoru; Sisido, Masahiko

2004-01-01

337

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

338

Drug delivery systems based on nucleic acid nanostructures.  

PubMed

The field of DNA nanotechnology has progressed rapidly in recent years and hence a large variety of 1D-, 2D- and 3D DNA nanostructures with various sizes, geometries and shapes is readily accessible. DNA-based nanoobjects are fabricated by straight forward design and self-assembly processes allowing the exact positioning of functional moieties and the integration of other materials. At the same time some of these nanosystems are characterized by a low toxicity profile. As a consequence, the use of these architectures in a biomedical context has been explored. In this review the progress and possibilities of pristine nucleic acid nanostructures and DNA hybrid materials for drug delivery will be discussed. For the latter class of structures, a distinction is made between carriers with an inorganic core composed of gold or silica and amphiphilic DNA block copolymers that exhibit a soft hydrophobic interior. PMID:23742878

de Vries, Jan Willem; Zhang, Feng; Herrmann, Andreas

2013-12-10

339

Imaging of nucleic acids with atomic force microscopy  

PubMed Central

Atomic force microscopy (AFM) is a key tool of nanotechnology with great importance in applications to DNA nanotechnology and to the recently emerging field of RNA nanotechnology. Advances in the methodology of AFM now enable reliable and reproducible imaging of DNA of various structures, topologies, and DNA and RNA nanostructures. These advances are reviewed here with emphasis on methods utilizing modification of mica to prepare the surfaces enabling reliable and reproducible imaging of DNA and RNA nanostructures. Since the AFM technology for DNA is more mature, AFM imaging of DNA is introduced in this review to provide experience and background for the improvement of AFM imaging of RNA. Examples of imaging different structures of RNA and DNA are discussed and illustrated. Special attention is given to the potential use of AFM to image the dynamics of nucleic acids at the nanometer scale. As such, we review recent advances with the use of time-lapse AFM.

Lyubchenko, Yuri L.; Shlyakhtenko, Luda S.; Ando, Toshio

2011-01-01

340

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

341

2011 Rita Schaffer Lecture: Nanoparticles for Intracellular Nucleic Acid Delivery  

PubMed Central

Nanoparticles are a promising technology for delivery of new types of therapeutics. A polymer library approach has allowed engineering of polymeric particles that are particularly effective for the delivery of DNA and siRNA to human cells. Certain chemical structural motifs, degradable linkages, hydrophobicity, and biophysical properties are key for successful intracellular delivery. Small differences to biomaterial structure, and especially the type of degradable linkage in the polymers, can be critical for successful delivery of siRNA vs. DNA. Furthermore, subtle changes to biomaterial structure can facilitate cell-type gene delivery specificity between human brain cancer cells and healthy cells as well as between human retinal endothelial cells and epithelial cells. These polymeric nanoparticles are effective for nucleic acid delivery in a broad range of human cell types and have applications to regenerative medicine, ophthalmology, and cancer among many other biomedical research areas.

Green, Jordan J.

2012-01-01

342

Three-arm nucleic acid junctions are flexible.  

PubMed

Nucleic acid junctions are stable analogs of branched DNA structures which occur transiently in living systems. We show here that junctions which contain three double helical arms can be enzymatically oligomerized, using conventional sticky-ended ligation procedures, to create larger complexes. The products consist of a series of linked junctions separated by 20 base pairs. Junction dimers are formed that have free termini only, whereas trimers and larger species are found to be both unclosed and cyclized. The formation of a series of macrocyclic products which, surprisingly, begins with trimers and tetramers indicates that this junction is flexible about a bending axis, and perhaps twist-wise as well. We have obtained the same results from three different 3-arm junctions, two in which the junction is flanked by a 3 Watson-Crick base pairs, and one in which a G-G base pair flanks the junction. PMID:3808954

Ma, R I; Kallenbach, N R; Sheardy, R D; Petrillo, M L; Seeman, N C

1986-12-22

343

Luminescent Probes for Ultrasensitive Detection of Nucleic Acids  

PubMed Central

Novel amino-reactive derivatives of lanthanide-based luminescent labels of enhanced brightness and metal retention were synthesized and used for the detection of complementary DNA oligonucleotides by molecular beacons. Time-resolved acquisition of the luminescent signal that occurs upon hybridization of the probe to the target enabled the avoidance of short-lived background fluorescence, markedly enhancing the sensitivity of detection, which was less than 1 pM. This value is about 50 to 100 times more sensitive than the level achieved with conventional fluorescence-based molecular beacons, and is 10 to 60 times more sensitive than previously reported for other lanthanide-based hybridization probes. These novel luminescent labels should significantly enhance the sensitivity of all type of nucleic acid hybridization probes, and could dramatically improve the detection limit of other biopolymers and small compounds that are used in a variety of biological applications.

Krasnoperov, Lev N.; Marras, Salvatore A.E.; Kozlov, Maxim; Wirpsza, Laura; Mustaev, Arkady

2010-01-01

344

Intriguing relations of interaction energy components in stacked nucleic acids  

NASA Astrophysics Data System (ADS)

Major components of the interaction energy that define several approximate levels starting from second order Möller-Plesset theory were studied for 58 stacked nucleic acid dimers. They included typical B-DNA and A-DNA structures, and selected published geometries. A survey of the various terms yields an unexpected correlation between the Pauli exchange and dispersion or correlation terms, which holds for each class of similar planar geometries and for various basis sets. The geometries that exhibit these correlations span a specific range of molecular overlaps when compared to a model benzene-pyridine stacked dimer. Also, the relationship between electrostatic interactions and MP2 stabilization energies reported earlier is confirmed and a prediction interval of practical relevance is estimated.

Langner, Karol M.; Sokalski, W. Andrzej; Leszczynski, J.

2007-09-01

345

Clinical applications of nucleic acid aptamers in cancer  

PubMed Central

Nucleic acid aptamers are small single-stranded DNA or RNA oligonucleotide segments, which bind to their targets with high affinity and specificity via unique three-dimensional structures. Aptamers are generated by an iterative in vitro selection process, termed as systematic evolution of ligands by exponential enrichment. Owing to their specificity, non-immunogenicity, non-toxicity, easily modified chemical structure and wide range of targets, aptamers appear to be ideal candidates for various clinical applications (diagnosis or treatment), such as cell detection, target diagnosis, molecular imaging and drug delivery. Several aptamers have entered the clinical pipeline for applications in diseases such as macular degeneration, coronary artery bypass graft surgery and various types of cancer. The aim of this review was to summarize and highlight the clinical applications of aptamers in cancer diagnosis and treatment.

PEI, XIAOYU; ZHANG, JUN; LIU, JIE

2014-01-01

346

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

347

Selective Nucleic Acid Capture with Shielded Covalent Probes  

PubMed Central

Nucleic acid probes are used for diverse applications in vitro, in situ, and in vivo. In any setting, their power is limited by imperfect selectivity (binding of undesired targets) and incomplete affinity (binding is reversible, and not all desired targets bound). These difficulties are fundamental, stemming from reliance on base pairing to provide both selectivity and affinity. Shielded covalent (SC) probes eliminate the longstanding trade-off between selectivity and durable target capture, achieving selectivity via programmable base pairing and molecular conformation change, and durable target capture via activatable covalent cross-linking. In pure and mixed samples, SC probes covalently capture complementary DNA or RNA oligo targets and reject two-nucleotide mismatched targets with near-quantitative yields at room temperature, achieving discrimination ratios of 2–3 orders of magnitude. Semiquantitative studies with full-length mRNA targets demonstrate selective covalent capture comparable to that for RNA oligo targets. Single-nucleotide DNA or RNA mismatches, including nearly isoenergetic RNA wobble pairs, can be efficiently rejected with discrimination ratios of 1–2 orders of magnitude. Covalent capture yields appear consistent with the thermodynamics of probe/target hybridization, facilitating rational probe design. If desired, cross-links can be reversed to release the target after capture. In contrast to existing probe chemistries, SC probes achieve the high sequence selectivity of a structured probe, yet durably retain their targets even under denaturing conditions. This previously incompatible combination of properties suggests diverse applications based on selective and stable binding of nucleic acid targets under conditions where base-pairing is disrupted (e.g., by stringent washes in vitro or in situ, or by enzymes in vivo).

2013-01-01

348

Up-converting phosphor reporters for nucleic acid microarrays.  

PubMed

An important application of robotically spotted DNA microarrays is the monitoring of RNA expression levels. A clear limitation of this technology is the relatively large amount of RNA that is required per hybridization as a result of low hybridization efficiency and limiting detection sensitivity provided by conventional fluorescent reporters. We have used a recently introduced luminescent reporter technology, called UPT (up-converting phosphor technology). Down-converting phosphors have been applied before to detect nucleic acids on filters using time-resolved fluorometry. The unique feature of the phosphor particles (size 0.4 microm) used here is that they emit visible light when illuminated with infrared (IR) light (980 nm) as a result of a phenomenon called up-conversion. Because neither support material of microarrays nor biomolecules possess up-conversion properties, an enhanced image contrast is expected when these nonfading phosphor particles are applied to detect nucleic acid hybrids on microarrays. Comparison of the UPT reporter to cyanin 5 (Cy5) in a low-complexity model system showed a two order of maginitude linear relationship between phosphor luminescence and target concentration and resulted in an excellent correlation between the two reporter systems for variable target concentrations (R2 = 0.95). However, UPT proved to be superior in sensitivity, even though a wide-field microscope equipped with a xenon lamp was used. This higher sensitivity was demonstrated by complementary DNA (cDNA) microarray hybridizations using cDNAs for housekeeping genes as probes and complex cDNA as target. These results suggest that a UPT reporter technology in combination with a dedicated IR laser array-scanner holds significant promise for various microarray applications. PMID:11231563

van De Rijke, F; Zijlmans, H; Li, S; Vail, T; Raap, A K; Niedbala, R S; Tanke, H J

2001-03-01

349

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.

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

2012-01-01

350

Seasonal changes in nucleic acids, amino acids and protein content in juvenile Norway lobster ( Nephrops norvegicus )  

Microsoft Academic Search

The objective of this study was to describe the seasonal variations in nucleic acid contents and amino acid profiles in the muscle of juvenile Nephrops norvegicus. RNA and protein contents, and RNA:protein and RNA:DNA ratios varied significantly between seasons, being highest in spring and lowest in autumn\\/winter ( PPP=0.05). In respect to protein-bound amino acid content (BAA), a significant increase

R. Rosa; M. L. Nunes

2003-01-01

351

Organic Acid Metabolism by Isolated Rhizobium japonicum Bacteroids  

PubMed Central

Rhizobium japonicum bacteroids isolated from soybean (Glycine max L.) nodules oxidized 14C-labeled succinate, pyruvate, and acetate in a manner consistent with operation of the tricarboxylic acid cycle and a partial glyoxylate cycle. Substrate carbon was incorporated into all major cellular components (cell wall + membrane, nucleic acids, and protein).

Stovall, Iris; Cole, Michael

1978-01-01

352

Suitability of an automated nucleic acid extractor (easyMAG) for use with hepatitis C virus and human immunodeficiency virus type 1 nucleic acid amplification testing.  

PubMed

Serological screening assays have greatly reduced, but not eliminated, the risk of transmission of viral infections by transfusion of blood and blood products. In addition, the 1999 regulation of the European Agency for the Evaluation of Medicinal Products requiring all plasma for fractionation to have tested negative for hepatitis C virus (HCV) RNA (CPMP/BWP/390/97, 1998) led many blood transfusion services to introduce nucleic acid amplification technology (NAT) to screen blood donations for HCV, and in some services for human immunodeficiency virus (HIV) and hepatitis B virus (HBV). BioMérieux's second-generation system, the NucliSENS easyMAG, was evaluated as a suitable platform for the automated extraction of nucleic acids for use with the existing SNBTS NAT assays. Two nucleic acid extraction protocols were examined, either lysis on the easyMAG (on board) or a 30-min pre-incubation of the sample with lysis buffer at 37 °C (off board). Off board lysis was found to extract nucleic acid more efficiently for both HCV and HIV NAT assays although the improvement was more marked with HIV. The 95% limit of detections (LODs) were 10.11 IU/ml (on board) and 7.21 IU/ml (off board) for HCV and 55.11 IU/ml (on board) and 34.13 (off board) for HIV. Using the more sensitive off board lysis, nucleic acid extraction specificity, robustness and reliability of the easyMAG were examined and over 10,000 Scottish blood donations (in 107 pools of 95 donations) were tested for HCV and HIV in parallel with the existing assay. The results indicate that the easyMAG is a suitable and flexible nucleic acid extraction system, providing high quality nucleic acids and a rapid response alternative to commercial, fully automated, approved blood screening platforms. PMID:21126541

Jarvis, L M; Mulligan, K; Dunsford, T H; McGowan, K; Petrik, J

2011-02-01

353

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

354

Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins  

PubMed Central

The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experiments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be ?10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are consistent with the primary function of Gag as a nucleic acid binding and packaging protein and the primary function of the NC proteins as nucleic acid chaperones. Also, our results suggest that NCp7's capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nucleic acid strand annealing by inducing electrostatic attraction between strands, likely optimize the fully processed NC protein to facilitate complex nucleic acid secondary structure rearrangements. In contrast, Gag's stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization.

Cruceanu, Margareta; Urbaneja, Maria A.; Hixson, Catherine V.; Johnson, Donald G.; Datta, Siddhartha A.; Fivash, Matthew J.; Stephen, Andrew G.; Fisher, Robert J.; Gorelick, Robert J.; Casas-Finet, Jose R.; Rein, Alan; Rouzina, Ioulia; Williams, Mark C.

2006-01-01

355

Nucleic Acid Sensors and Type I Interferon Production in Systemic Lupus Erythematosus  

PubMed Central

The characteristic serologic feature of systemic lupus erythematosus (SLE) is autoantibodies against one’s own nucleic acid or nucleic acid-binding proteins – DNA and RNA-binding nuclear proteins. Circulating autoantibodies can deposit in the tissue, causing inflammation and production of cytokines such as type 1 interferon (IFN). Investigations in human patients and animal models have implicated environmental as well as genetic factors in the biology of the SLE autoimmune response. Viral/Bacterial nucleic acid is a potent stimulant of innate immunity by both toll-like receptor (TLR) and non-TLR signaling cascades. Additionally, foreign DNA may act as an immunogen to drive an antigen-specific antibody response. Self nucleic acid is normally restricted to the nucleus or the mitochondria, away from the DNA/RNA sensors, and mechanisms exist to differentiate between foreign and self nucleic acid. In normal immunity, a diverse range of DNA and RNA sensors in different cell types form a dynamic and integrated molecular network to prevent viral infection. In SLE, pathologic activation of these sensors occurs via immune complexes consisting of autoantibodies bound to DNA or to nucleic acid-protein complexes. In this review, we will discuss recent studies outlining how mismanaged nucleic acid sensing networks promote autoimmunity and result in the over-production of type I IFN. This information is critical for improving therapeutic strategies for SLE disease.

Shrivastav, Meena; Niewold, Timothy B.

2013-01-01

356

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.

Brencicova, Eva; Diebold, Sandra S.

2013-01-01

357

Effects of Phosfon-S on Nucleic Acid Metabolism in Pisum sativum Alaska  

PubMed Central

Phosfon-S, a substance which inhibits stem elongation, alters nucleic acid metabolism in Pisum sativum Alaska. Methylated albumin kieselguhr (MAK) columns were used to fractionate 32P-labeled nucleic acids. Phosfon-S treatment of the plants resulted in a decrease in soluble RNA and an increase in ribosomal RNA. Specific activities of the various nucleic acid fractions were lower as a result of treatment. The nucleic acids from treated tissues were more resistant to RNase degradation, and endogenous RNase activity was lower in treated tissues. When RNase treated nucleic acids were fractionated on MAK columns, the DNA-RNA fractions from treated plants had a higher specific activity than that of the control, which was not true before nuclease treatment. Spectrophotometric examination of this fraction revealed a difference in absorption spectra, possibly indicating a Phosfon-S nucleic acid complex. It is suggested that these alterations in nucleic acid metabolism could in turn alter a wide variety of metabolic processes, resulting in retarded growth.

Brook, Judith; West, S. H.; Anthony, D. S.

1967-01-01

358

Detection of Salmonella invA by isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) in Zambia  

Microsoft Academic Search

The isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) is a new isothermal DNA amplification method composed of exo Bca DNA polymerase, RNaseH and DNA–RNA chimeric primers. We detected invA of Salmonella from chicken carcasses, egg yolk and cattle fecal samples. Fifty-three of 59 isolates were invA-positive in ICAN-chromatostrip detection. The result was consistent with those obtained by standard

Emiko Isogai; Chitwambi Makungu; John Yabe; Patson Sinkala; Andrew Nambota; Hiroshi Isogai; Hideto Fukushi; Manda Silungwe; Charles Mubita; Michelo Syakalima; Bernard Mudenda Hang'ombe; Shunji Kozaki; Jun Yasuda

2005-01-01

359

MS analysis of nucleic acids in the post-genomic era  

PubMed Central

Alternative approaches complementing the existing technologies for analysis of nucleic acids and their assemblies are necessary to take on the new challenges posed by the post-genomic era. The versatility of MS in biopolymer analysis and its ability to reach beyond sequence information are the basis of ever expanding applications aimed at the elucidation of nucleic acid structure-function relationships. This Feature summarizes the current state of MS-based approaches devised to overcome the limitations of traditional techniques and to advance different facets of nucleic acids research.

Fabris, D.

2011-01-01

360

Strategies for automated sample preparation, nucleic acid purification, and concentration of low-target-number nucleic acids in environmental and food processing samples  

NASA Astrophysics Data System (ADS)

The purpose of this work is to develop a rapid, automated system for nucleic acid purification and concentration from environmental and food processing samples. Our current approach involves off-line filtration and cell lysis (ballistic disintegration) functions in appropriate buffers followed by automated nucleic acid capture and purification on renewable affinity matrix microcolumns. Physical cell lysis and renewable affinity microcolumns eliminate the need for toxic organic solvents, enzyme digestions or other time- consuming sample manipulations. Within the renewable affinity microcolumn, we have examined nucleic acid capture and purification efficiency with various microbead matrices (glass, polymer, paramagnetic), surface derivitization (sequence-specific capture oligonucleotides or peptide nucleic acids), and DNA target size and concentration under variable solution conditions and temperatures. Results will be presented comparing automated system performance relative to benchtop procedures for both clean (pure DNA from a laboratory culture) and environmental (soil extract) samples, including results which demonstrate 8 minute purification and elution of low-copy nucleic acid targets from a crude soil extract in a form suitable for PCR or microarray-based detectors. Future research will involve the development of improved affinity reagents and complete system integration, including upstream cell concentration and cell lysis functions and downstream, gene-based detectors. Results of this research will ultimately lead to improved processes and instrumentation for on-line, automated monitors for pathogenic micro-organisms in food, water, air, and soil samples.

Bruckner-Lea, Cynthia J.; Holman, David A.; Schuck, Beatrice L.; Brockman, Fred J.; Chandler, Darrell P.

1999-01-01

361

Interaction of cetylpyridine bromide with nucleic acids and determination of nucleic acids at nanogram levels based on the enhancement of resonance Rayleigh light scattering  

NASA Astrophysics Data System (ADS)

Resonance Rayleigh light scattering (RRLS) spectra of cetylpyridine bromide (CPB)-nucleic acid system and their analytical application have been first studied. The effective factors and optimum conditions of the reaction have been investigated. After CPB and nucleic acid are mixed together, a new absorption peak located at 300 nm appeared, which is due to the formation of new ion associate of CPB-nucleic acid. The new associate can result in two apparent RRLS peaks at 310-400 and 460-480 nm. The RRLS peak of the corrected spectra located at 290-350 nm, which indicate that the RRLS is originated from the absorption of CPB-nucleic acid associate. The peak at 460-480 nm disappears in the corrected RRLS spectra, which indicated that this peak is originated from the strong line emission of the Xe lamp. Under the optimum conditions, the enhanced intensity of RRLS is proportional to the concentration of nucleic acid in the range of 5.0×10 -9-5.0×10 -5 g ml -1 for calf thymus DNA (ctDNA), 1.0×10 -8-4.0×10 -5 g ml -1 for fish sperm DNA (fsDNA) and 1.0×10 -8-5.0×10 -5 g ml -1 for yeast RNA (yRNA). The detection limits ( S/ N=3) are 4.3, 8.7 and 7.4 ng ml -1, respectively. Synthetic samples were determined satisfactorily.

Liu, Rutao; Yang, Jinghe; Wu, Xia

2002-07-01

362

Reaction of urethane with nucleic acids in vivo  

PubMed Central

1. [1-14C]Ethyl carbamate, ethyl [carboxy-14C]carbamate, [1-14C]ethanol and sodium hydrogen [14C]carbonate were injected intraperitoneally into C57 mice, and nucleic acids and proteins were separated from the liver and lungs with phenol as described by Kirby (1956). 2. Chromatographic analysis of the hydrolytic products of the urethane-labelled RNA showed the presence of a single radioactive compound differing in behaviour from the major pyrimidine nucleotides and purines. 3. The products from RNA labelled by [1-14C]ethyl carbamate or ethyl [carboxy-14C]carbamate appeared chromatographically identical but could not be detected in the RNA of mice given [1-14C]ethanol or sodium hydrogen [14C]-carbonate. 4. The labelled product appeared to be the ethyl ester of cytosine-5-carboxylic acid formed by the reaction of urethane with RNA in vivo. 5. A direct reaction between labelled urethane or the labelled metabolite of urethane, [1-3H]-ethyl N-hydroxycarbamate, and RNA was not detected.

Boyland, E.; Williams, K.

1969-01-01

363

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

364

Are isolated anti-HBc blood donors in high risk group? The detection of HBV DNA in isolated anti-HBc cases with nucleic acid amplification test (NAT) based on transcription-mediated amplification (TMA) and HBV discrimination  

Microsoft Academic Search

AimHepatitis B virus (HBV) can be transmitted by blood transfusions even so using serological tests having high sensitivity and specificity. We aimed to detect HBV DNA in isolated Anti-HBc donors and show if they have transfusion risk or not.

Hüsnü Altunay; Erdogan Kosan; Ilhan Birinci; Armagan Aksoy; Kaan Kirali; Suat Saribas; Mustafa Aslan; Pelin Yuksel; Esra Alan; Osman Sadi Yenen; Bekir Kocazeybek

2010-01-01

365

Stability of the Nucleic Acids of L Cells After Infection with the Meningopneumonitis Agent  

PubMed Central

The stability of host nucleic acids in L cells infected with Chlamydia psittaci (strain meningopneumonitis) was studied. The L cells were prelabeled with either 32P-orthophosphate, 3H-uridine, or 3H-thymidine. After infection, the redistribution of each label among the different fractions of host and parasite was quantitatively determined and compared. There were no signs of accelerated degradation of host nucleic acid as the consequence of meningopneumonitis infection. Comparison of the specific activities of the meningopneumonitis nucleic acids with that of the acid-soluble fraction of host cell cytoplasm suggested that the major source of precursors for parasite nucleic acid synthesis was the common cytoplasmic pool of the infected host cell.

Lin, Hsiu-San

1968-01-01

366

Cell-free nucleic acids as potential markers for preeclampsia.  

PubMed

Preeclampsia is one of the leading causes of maternal and fetal/neonatal mortality and morbidity worldwide. Therefore, widely applicable and affordable tests are needed to make an early diagnosis before the occurrence of the clinical symptoms. Circulating cell-free nucleic acids in plasma and serum are novel biomarkers with promising clinical applications in different medical fields, including prenatal diagnosis. Quantitative changes of cell-free fetal (cff)DNA in maternal plasma as an indicator for impending preeclampsia have been reported in different studies, using real-time quantitative PCR for the male-specific SRY or DYS 14 loci. In case of early onset preeclampsia, elevated levels may be already seen in the first trimester. The increased levels of cffDNA before the onset of symptoms may be due to hypoxia/reoxygenation within the intervillous space leading to tissue oxidative stress and increased placental apoptosis and necrosis. In addition to the evidence for increased shedding of cffDNA into the maternal circulation, there is also evidence for reduced renal clearance of cffDNA in preeclampsia. As the amount of fetal DNA is currently determined by quantifying Y-chromosome specific sequences, alternative approaches such as the measurement of total cell-free DNA or the use of gender-independent fetal epigenetic markers, such as DNA methylation, offer a promising alternative. Cell-free RNA of placental origin might be another potentially useful biomarker for screening and diagnosis of preeclampsia in clinical practice. Fetal RNA is associated with subcellular placental particles that protect it from degradation. Its levels are ten-fold higher in pregnant women with preeclampsia compared to controls. In conclusion, through the use of gender-independent sequences, the universal incorporation of fetal nucleic acids into routine obstetric care and into screening or diagnostic settings using combined markers may soon become a reality. Effort has now to be put into the establishment of standardized and simplified protocols for the analysis of these biomarkers in a clinical setting. PMID:21257079

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

2011-02-01

367

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.

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

368

Nucleic Acid Aptamers as Potential Therapeutic and Diagnostic Agents for Lymphoma  

PubMed Central

Lymphomas are cancers that arise from white blood cells and usually present as solid tumors. Treatment of lymphoma often involves chemotherapy, and can also include radiotherapy and/or bone marrow transplantation. There is an un-questioned need for more effective therapies and diagnostic tool for lymphoma. Aptamers are single stranded DNA or RNA oligonucleotides whose three-dimensional structures are dictated by their sequences. The immense diversity in function and structure of nucleic acids enable numerous aptamers to be generated through an iterative in vitro selection technique known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers have several biochemical properties that make them attractive tools for use as potential diagnostic and pharmacologic agents. Isolated aptamers may directly inhibit the function of target proteins, or they can also be formulated for use as delivery agents for other therapeutic or imaging cargoes. More complex aptamer identification methods, using whole cancer cells (Cell-SELEX), may identify novel targets and aptamers to affect them. This review focuses on recent advances in the use of nucleic acid aptamers as diagnostic and therapeutic agents and as targeted delivery carriers that are relevant to lymphoma. Some representative examples are also discussed.

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

2014-01-01

369

Nucleic Acids and Polypeptides Useful for Diagnosing and Treating Complications of Pregnancy.  

National Technical Information Service (NTIS)

Disclosed herein are methods for diagnosing or treating pregnancy related hypertensive disorders that include the use of a polypeptide or a nucleic acid encoding a polypeptide selected from the following: follistatin related protein, interleukin 8, inhibi...

S. A. Karumanchi V. P. Sukhatme

2005-01-01

370

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”).

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

1982-01-01

371

Nucleic Acid Intercalators Suppress Strand Cyclization in Model Prebiotic Polymerization Reactions  

NASA Astrophysics Data System (ADS)

We show that base pair intercalation by small molecules, which stabilizes and rigidifies nucleic acid duplexes, reduces strand cyclization, allowing for chemical ligation of tetranucleotides into duplex polymers of at least 100 bp in length.

Horowitz, E. D.; Engelhart, A. E.; Hud, N. V.

2010-04-01

372

On Nucleic Acids Accumulating under the Influence of Streptomycin in Escherichia Coli.  

National Technical Information Service (NTIS)

We have discovered (Bacteriol. Proc., p. 131, 1959) that addition of streptomycin (SM) to logphase cultures of E. coli, Gratia, is followed by cessation of protein synthesis while nucleic acid synthesis continues for approximately one generation time to i...

J. Ciak F. E. Hahn

1964-01-01

373

Determination of nucleic acids at nanogram level using resonance light scattering technique with Congo Red  

NASA Astrophysics Data System (ADS)

Based on the enhancement of the resonance light scattering (RLS) of Congo Red (CR) by nucleic acid, a new quantitative method for nucleic acid is developed. In the Tris-HCl buffer (pH 10.5), the weak light scattering of CR is greatly enhanced by addition of nucleic acid and CTMAB, the maximum peak is at 560 nm and the enhanced intensity of RLS is in proportion to the concentration of nucleic acid. The linear range is 1.0×10 -9 to 1.0×10 -6 g ml -1, 7.5×10 -8 to 1.0×10 -6 g ml -1 and 7.5×10 -8 to 2.5×10 -6 g ml -1 for herring sperm DNA, calf thymus DNA and yeast RNA, and the detection limits are 0.019, 0.89 and 1.2 ng ml -1 ( S/ N = 3), respectively. Actual biological samples were satisfactorily determined.

Wu, Xia; Wang, Yuebo; Wang, Minqin; Sun, Shuna; Yang, Jinghe; Luan, Yuxia

2005-01-01

374

Extraction of Total Nucleic Acids From Ticks for the Detection of Bacterial and Viral Pathogens  

PubMed Central

Ticks harbor numerous bacterial, protozoal, and viral pathogens that can cause serious infections in humans and domestic animals. Active surveillance of the tick vector can provide insight into the frequency and distribution of important pathogens in the environment. Nucleic-acid based detection of tick-borne bacterial, protozoan, and viral pathogens requires the extraction of both DNA and RNA (total nucleic acids) from ticks. Traditional methods for nucleic acid extraction are limited to extraction of either DNA or the RNA from a sample. Here we present a simple bead-beating based protocol for extraction of DNA and RNA from a single tick and show detection of Borrelia burgdorferi and Powassan virus from individual, infected Ixodes scapularis ticks. We determined expected yields for total nucleic acids by this protocol for a variety of adult tick species. The method is applicable to a variety of arthropod vectors, including fleas and mosquitoes, and was partially automated on a liquid handling robot.

Crowder, Chris D.; Rounds, Megan A.; Phillipson, Curtis A.; Picuri, John M.; Matthews, Heather E.; Halverson, Justina; Schutzer, Steven E.; Ecker, David J.; Eshoo, Mark W.

2010-01-01

375

Fluorescent imaging and quantitation of solid support-bound nucleic acids  

NASA Astrophysics Data System (ADS)

Recent advances in molecular biology have enabled the deposition of nucleic acids on solid phases to form arrays of oligonucleotides. Such arrays are being applied in milli to nanoscale molecular and biochemical analysis such as genetic mutation detection, gene expression quantitation and DNA sequencing using so-called DNA chip arrays. A major obstacle to continue use of such arrays is detection and analysis of the arrayed nucleic acids, nucleic acids, nucleic acids' targets that bind via hybridization to the arrays and the products of array-based biochemical reactions. We report here on the design and utility of our experimental set-up for analysis of surface bound, arrayed oligonucleotides, and our experience in detection and quantitation of multiple fluorescent labels bound to the surface through attachment, hybridization, and arrayed primer extension.

Bogdanov, Valery L.; Rogers, Yu-Hui; Boyce-Jacino, Michael

1997-05-01

376

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

377

Rare-earth-cation-induced change in the cholesteric twisting of neighboring nucleic acid molecules  

SciTech Connect

Certain physicochemical characteristics of particles of the cholesteric liquid-crystal dispersions of complexes of double-stranded nucleic acids with rare earth elements have been determined. It is shown for the first time that the binding of the rare earth cations to linear nucleic acid molecules ordered in the structure of particles of the cholesteric liquid crystal dispersions is accompanied not only by amplification of the abnormal band in the circular dichroism spectrum, but also by the disappearance of the characteristic maximum on the X-ray scattering curves for small angles. The (cholesteric 1-cholesteric 2) transition induced by rare earth cations is an example of the operation of a microscopic machine consisting of spatially ordered nucleic acid molecules. Particles of the cholesteric liquid crystal dispersions of nucleic acid complexes with rare earth elements hold the abnormal optical properties for a long time.

Yevdokimov, Yu. M., E-mail: yevdokim@eimb.ru; Salyanov, V. I.; Kondrashina, O. V. [Russian Academy of Sciences, Engelhardt Institute of Molecular Biology (Russian Federation); Gasanov, A. A. [Giredmet Research and Design Institute (Russian Federation); Shtykova, E. V.; Dembo, K. A. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

2007-03-15

378

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

PubMed

Veterinary pathology of infectious, particularly viral, and neoplastic diseases has advanced significantly with the advent of newer molecular methodologies that can detect nucleic acid of infectious agents within microscopic lesions, differentiate neoplastic from nonneoplastic cells, or determine the suitability of a targeted therapy by detecting specific mutations in certain cancers. Polymerase chain reaction-based amplification of DNA or RNA and in situ hybridization are currently the most commonly used methods for nucleic acid detection. In contrast, the main methodology used for protein detection within microscopic lesions is immunohistochemistry. Other methods that allow for analysis of nucleic acids within a particular cell type or individual cells, such as laser capture microdissection, are also available in some laboratories. This review gives an overview of the factors that influence the accurate analysis of nucleic acids in formalin-fixed tissues, as well as of different approaches to detect such targets. PMID:24129897

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

2014-01-01

379

Rapid genotyping using pyrene-perylene locked nucleic acid complexes  

PubMed Central

We have developed an assay for single strand DNA and RNA detection which is based on novel pyrene?perylene FRET pairs attached to short LNA/DNA probes. The assay is based on ratiometric emission upon binding of target DNA/RNA by three combinations of fluorescent LNA/DNA reporter strands. Specific geometry of the pyrene fluorophore attached to the 2?-amino group of 2?-amino-LNA in position 4 allows for the first time to efficiently utilize dipole?dipole orientation parameter for sensing of single-nucleotide polymorphisms (SNPs) in nucleic acid targets by FRET. Using novel probes, SNP detection is achieved with advantages of large Stokes shift (115 nm), high fluorescence quantum yields and low limit of target detection values (< 5 nM). Rapid and accurate genotyping of highly polymorphic HIV Pol cDNA and RNA fragments performed herein proves the possibility for broad application of the novel pyrene?perylene FRET pairs, e.g., in imaging and clinical diagnostics.

Kumar, T. Santhosh; Myznikova, Anna; Samokhina, Evgeniya; Astakhova, Irina Kira

2013-01-01

380

Solution influence on biomolecular equilibria - Nucleic acid base associations  

NASA Technical Reports Server (NTRS)

Various attempts to construct an understanding of the influence of solution environment on biomolecular equilibria at the molecular level using computer simulation are discussed. First, the application of the formal statistical thermodynamic program for investigating biomolecular equilibria in solution is presented, addressing modeling and conceptual simplications such as perturbative methods, long-range interaction approximations, surface thermodynamics, and hydration shell. Then, Monte Carlo calculations on the associations of nucleic acid bases in both polar and nonpolar solvents such as water and carbon tetrachloride are carried out. The solvent contribution to the enthalpy of base association is positive (destabilizing) in both polar and nonpolar solvents while negative enthalpies for stacked complexes are obtained only when the solute-solute in vacuo energy is added to the total energy. The release upon association of solvent molecules from the first hydration layer around a solute to the bulk is accompanied by an increase in solute-solvent energy and decrease in solvent-solvent energy. The techniques presented are expectd to displace less molecular and more heuristic modeling of biomolecular equilibria in solution.

Pohorille, A.; Pratt, L. R.; Burt, S. K.; Macelroy, R. D.

1984-01-01

381

Nucleic acid dyes for detection of apoptosis in live cells.  

PubMed

Apoptotic thymocytes were found to be much dimmer than normal thymocytes when stained with several nucleic acid dyes. These dyes provide a quick and simple assay for apoptosis which works for live cells and does not require a UV laser. The collection of dyes giving this staining pattern includes reagents suitable for use in either the FL1, FL2, or FL3 channel of a standard FACScan. Cells identified by these reagents were identical to apoptotic thymocytes defined by several widely used criteria: (i) rapid uptake of Hoechst 33342 but exclusion of propidium iodide, (ii) merocyanin 540 bright, and (iii) sub-G1 DNA content when permeabilized in a buffer that elutes fragmented DNA. In addition, L3T4/Thy-1 dim thymocytes were included in the dyc dim population. The standard Hoechst 33342 and merocyanin 540 assays were not able to separate the normal and apoptotic populations in HL-60 cells treated with camptothecin. However, the dyes SYTO-16 and LDS-751 both gave adequate differentiation of apoptotic from nonapoptotic cells in this model system. Some of these dyes also emit very little in other fluorescence channels of the flow cytometer and can be used in multicolor assays on cytometers equipped with only a single argon-ion laser. PMID:8582249

Frey, T

1995-11-01

382

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

383

Membrane Protected Apoptotic Trophoblast Microparticles Contain Nucleic Acids  

PubMed Central

Microparticles (MPs) that circulate in blood may be a source of DNA for molecular analyses, including prenatal genetic diagnoses. Because MPs are heterogeneous in nature, however, further characterization is important before use in clinical settings. One key question is whether DNA is either bound to aggregates of blood proteins and lipid micelles or intrinsically associated with MPs from dying cells. To test the latter hypothesis, we asked whether MPs derived in vitro from dying cells were similar to those in maternal plasma. JEG-3 cells model extravillous trophoblasts, which predominate during the first trimester of pregnancy when prenatal diagnosis is most relevant. MPs were derived from apoptosis and increased over 48 hours. Compared with necrotic MPs, DNA in apoptotic MPs was more fragmented and resistant to plasma DNases. Membrane-specific dyes indicated that apoptotic MPs had more membranous material, which protects nucleic acids, including RNA. Flow cytometry showed that MPs derived from dying cells displayed light scatter and DNA staining similar to MPs found in maternal plasma. Quantification of maternal MPs using characteristics defined by MPs generated in vitro revealed a significant increase of DNA+ MPs in the plasma of women with preeclampsia compared with plasma from women with normal pregnancies. Apoptotic MPs are therefore a likely source of stable DNA that could be enriched for both early genetic diagnosis and monitoring of pathological pregnancies.

Orozco, Aaron F.; Jorgez, Carolina J.; Horne, Cassandra; Marquez-Do, Deborah A.; Chapman, Matthew R.; Rodgers, John R.; Bischoff, Farideh Z.; Lewis, Dorothy E.

2008-01-01

384

Design of antisense oligonucleotides stabilized by locked nucleic acids  

PubMed Central

The design of antisense oligonucleotides containing locked nucleic acids (LNA) was optimized and compared to intensively studied DNA oligonucleotides, phosphorothioates and 2?-O-methyl gapmers. In contradiction to the literature, a stretch of seven or eight DNA monomers in the center of a chimeric DNA/LNA oligonucleotide is necessary for full activation of RNase H to cleave the target RNA. For 2?-O-methyl gapmers a stretch of six DNA monomers is sufficient to recruit RNase H. Compared to the 18mer DNA the oligonucleotides containing LNA have an increased melting temperature of 1.5–4°C per LNA depending on the positions of the modified residues. 2?-O-methyl nucleotides increase the Tm by only <1°C per modification and the Tm of the phosphorothioate is reduced. The efficiency of an oligonucleotide in supporting RNase H cleavage correlates with its affinity for the target RNA, i.e. LNA > 2?-O-methyl > DNA > phosphorothioate. Three LNAs at each end of the oligonucleotide are sufficient to stabilize the oligonucleotide in human serum 10-fold compared to an unmodified oligodeoxynucleotide (from t1/2 = ?1.5 h to t1/2 = ~15 h). These chimeric LNA/DNA oligonucleotides are more stable than isosequential phosphorothioates and 2?-O-methyl gapmers, which have half-lives of 10 and 12 h, respectively.

Kurreck, Jens; Wyszko, Eliza; Gillen, Clemens; Erdmann, Volker A.

2002-01-01

385

Ion trap collision-induced dissociation of locked nucleic acids.  

PubMed

Gas-phase dissociation of model locked nucleic acid (LNA) oligonucleotides and functional LNA-DNA chimeras have been investigated as a function of precursor ion charge state using ion trap collision-induced dissociation (CID). For the model LNA 5 and 8 mer, containing all four LNA monomers in the sequence, cleavage of all backbone bonds, generating a/w-, b/x-, c/y-, and d/z-ions, was observed with no significant preference at lower charge states. Base loss ions, except loss of thymine, from the cleavage of N-glycosidic bonds were also present. In general, complete sequence coverage was achieved in all charge states. For the two LNA-DNA chimeras, however, dramatic differences in the relative contributions of the competing dissociation channels were observed among different precursor ion charge states. At lower charge states, sequence information limited to the a-Base/w-fragment ions from cleavage of the 3'C-O bond of DNA nucleotides, except thymidine (dT), was acquired from CID of both the LNA gapmer and mixmer ions. On the other hand, extensive fragmentation from various dissociation channels was observed from post-ion/ion ion trap CID of the higher charge state ions of both LNA-DNA chimeras. This report demonstrates that tandem mass spectrometry is effective in the sequence characterization of LNA oligonucleotides and LNA-DNA chimeric therapeutics. PMID:19854063

Huang, Teng-yi; Kharlamova, Anastasia; McLuckey, Scott A

2010-01-01

386

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.

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

2013-01-01

387

Linear and nonlinear optical properties of nucleic acid bases  

NASA Astrophysics Data System (ADS)

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

Alparone, Andrea

2013-01-01

388

Charge-Neutral Morpholino Microarrays for Nucleic Acid Analysis  

PubMed Central

A principal challenge in microarray experiments is to facilitate hybridization between probe strands on the array with complementary target strands from solution while suppressing any competing interactions that the probes and targets may experience. Synthetic DNA analogs, whose hybridization to targets can exhibit qualitatively different dependence on experimental conditions than for nucleic acid probes, open up an attractive alternative for improving selectivity of array hybridization. Morpholinos (MOs), a class of uncharged DNA analogs, are investigated as microarray probes instead of DNA. Morpholino microarrays were fabricated by contact printing of amino-modified probes onto aldehyde slides. In addition to covalent immobilization, MOs were found to efficiently immobilize through physical adsorption; such physically adsorbed probes could be removed by post-printing washes with surfactant solutions. Hybridization of double-stranded DNA targets to MO microarrays revealed a hybridization maximum at intermediate ionic strengths. The decline in hybridization at lower ionic strengths was attributed to an electrostatic barrier accumulated from hybridized DNA targets, while at higher ionic strengths it was attributed to stabilization of target secondary structure in solution. These trends, which illustrate ionic strength tuning of forming on-array relative to solution secondary structure, were supported by a stability analysis of MO/DNA and DNA/DNA duplexes in solution.

Qiao, Wanqiong; Kalachikov, Sergey; Liu, Yatao; Levicky, Rastislav

2012-01-01

389

A microfluidic biosensor based on nucleic acid sequence recognition.  

PubMed

The development of a generic semi-disposable microfluidic biosensor for the highly sensitive detection of pathogens via their nucleic acid sequences is presented in this paper. Disposable microchannels with defined areas for capture and detection of target pathogen RNA sequence were created in polydimethylsiloxane (PDMS) and mounted onto a reusable polymethylmethacrylate (PMMA) stand. Two different DNA probes complementary to unique sequences on the target pathogen RNA serve as the biorecognition elements. For signal generation and amplification, one probe is coupled to dye encapsulated liposomes while the second probe is coupled to superparamagnetic beads for target immobilization. The probes hybridize to target RNA and the liposome-target-bead complex is subsequently captured on a magnet. The amount of liposomes captured correlates directly to the concentration of target sequence and is quantified using a fluorescence microscope. Dengue fever virus serotype 3 sequences and probes were used as a model analyte system to test the sensor. Probe binding and target capture conditions were optimized for sensitivity resulting in a detection limit of as little as 10 amol microL(-1) (10 pmol L(-1)). Future biosensors will be designed to incorporate a mixer and substitute the fluorescence detection with an electrochemical detection technique to provide a truly portable microbiosensor system. PMID:12830353

Kwakye, Sylvia; Baeumner, Antje

2003-08-01

390

To build a virus on a nucleic acid substrate.  

PubMed

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

2013-04-01

391

Proposed Ancestors of Phage Nucleic Acid Packaging Motors (and Cells)  

PubMed Central

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

Serwer, Philip

2011-01-01

392

ATP binding modulates the nucleic acid affinity of hepatitis C virus helicase.  

PubMed

The helicase of hepatitis C virus (HCV) unwinds nucleic acid using the energy of ATP hydrolysis. The ATPase cycle is believed to induce protein conformational changes to drive helicase translocation along the length of the nucleic acid. We have investigated the energetics of nucleic acid binding by HCV helicase to understand how the nucleotide ligation state of the helicase dictates the conformation of its nucleic acid binding site. Because most of the nucleotide ligation states of the helicase are transient due to rapid ATP hydrolysis, several compounds were analyzed to find an efficient unhydrolyzable ATP analog. We found that the beta-gamma methylene/amine analogs of ATP, ATPgammaS, or [AlF4]ADP were not effective in inhibiting the ATPase activity of HCV helicase. On the other hand, [BeF3]ADP was found to be a potent inhibitor of the ATPase activity, and it binds tightly to HCV helicase with a 1:1 stoichiometry. Equilibrium binding studies showed that HCV helicase binds single-stranded nucleic acid with a high affinity in the absence of ATP or in the presence of ADP. Upon binding to the ATP analog, a 100-fold reduction in affinity for ssDNA was observed. The reduction in affinity was also observed in duplex DNA with 3' single-stranded tail and in RNA but not in duplex DNA. The results of this study indicate that the nucleic acid binding site of HCV helicase is allosterically modulated by the ATPase reaction. The binding energy of ATP is used to bring HCV helicase out of a tightly bound state to facilitate translocation, whereas ATP hydrolysis and product release steps promote tight rebinding of the helicase to the nucleic acid. On the basis of these results we propose a Brownian motor model for unidirectional translocation of HCV helicase along the nucleic acid length. PMID:12660239

Levin, Mikhail K; Gurjar, Madhura M; Patel, Smita S

2003-06-27

393

UV and visible-excited fluorescence of nucleic acids separated by capillary electrophoresis  

Microsoft Academic Search

UV- and visible-excited fluorescence detection strategies were compared for nucleic acids separated by capillary electrophoresis (CE). A dual-polymer sieving matrix consisting of hydroxypropylmethylcellulose and poly(vinylpyrrolidone) was used to separate DNA fragments from a 100-base pair ladder and RNA from individual cells. Two nucleic acid dyes, SYBR Gold and SYBR Green I, were evaluated for their performance at both UV (275

Jennifer L Zabzdyr; Sheri J Lillard

2001-01-01

394

5[prime] to 3[prime] nucleic acid synthesis using 3[prime]-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[prime] to 3[prime] nucleic acid synthesis. The method can be used to prepare arrays of oligomers bound to a support via their 5[prime] 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, M.C.; Shuey, S.W.; Bradley, J.C.

1999-06-01

395

Locked Nucleic Acid: High-Affinity Targeting of Complementary RNA for RNomics  

Microsoft Academic Search

Locked nucleic acid (LNA) is a nucleic acid analog containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA-mimicking sugar conformation. This conformational restriction is translated into unprecedented hybridization affinity towards complementary single-stranded RNA molecules. That makes fully modified LNAs, LNA\\/DNA mixmers, or LNA\\/RNA mixmers uniquely suited for mimicking RNA structures and for RNA

S. Kauppinen; B. Vester; J. Wengel

396

Does Water Play a Structural Role in the Folding of Small Nucleic Acids?  

Microsoft Academic Search

Nucleic acid structure and dynamics are known to be closely coupled to local environmental conditions and, in particular, to the ionic character of the solvent. Here we consider what role the discrete properties of water and ions play in the collapse and folding of small nucleic acids. We study the folding of an experimentally well-characterized RNA hairpin-loop motif (sequence 5?-GGGC[GCAA]GCCU-3?)

Eric J. Sorin; Young Min Rhee; Vijay S. Pande

2005-01-01

397

Prediction of interactiveness of proteins and nucleic acids based on feature selections  

Microsoft Academic Search

It is important to identify which proteins can interact with nucleic acids for the purpose of protein annotation, since interactions\\u000a between nucleic acids and proteins involve in numerous cellular processes such as replication, transcription, splicing, and\\u000a DNA repair. This research tries to identify proteins that can interact with DNA, RNA, and rRNA, respectively. mRMR (Minimum\\u000a redundancy and maximum relevance), with

YouLang Yuan; XiaoHe Shi; XinLei Li; WenCong Lu; YuDong Cai; Lei Gu; Liang Liu; MinJie Li; XiangYin Kong; Meng Xing

2010-01-01

398

Prospects for nucleic acid-based therapeutics against hepatitis C virus  

PubMed Central

In this review, we discuss recent advances in nucleic acid-based therapeutic technologies that target hepatitis C virus (HCV) infection. Because the HCV genome is present exclusively in RNA form during replication, various nucleic acid-based therapeutic approaches targeting the HCV genome, such as ribozymes, aptamers, siRNAs, and antisense oligonucleotides, have been suggested as potential tools against HCV. Nucleic acids are potentially immunogenic and typically require a delivery tool to be utilized as therapeutics. These limitations have hampered the clinical development of nucleic acid-based therapeutics. However, despite these limitations, nucleic acid-based therapeutics has clinical value due to their great specificity, easy and large-scale synthesis with chemical methods, and pharmaceutical flexibility. Moreover, nucleic acid therapeutics are expected to broaden the range of targetable molecules essential for the HCV replication cycle, and therefore they may prove to be more effective than existing therapeutics, such as interferon-? and ribavirin combination therapy. This review focuses on the current status and future prospects of ribozymes, aptamers, siRNAs, and antisense oligonucleotides as therapeutic reagents against HCV.

Lee, Chang Ho; Kim, Ji Hyun; Lee, Seong-Wook

2013-01-01

399

Immunoenzymatic techniques applied to the specific detection of nucleic acids. A review.  

PubMed

Numerous enzymatic and chemical methods are now available for the preparation of non-radioactive nucleic acid probes. Labels, such as enzymes, fluorophores, lumiphores can be attached to the nucleic acid probe either by covalent bonds (direct labelling) or by biospecific recognition after hybridization (indirect labelling). The principle of the latter method is based on the use of a hapten-labelled nucleic acid probe which is generally detected by an immunoenzymatic assay. Indirect labelling has several advantages: this procedure uses multienzyme complexes to increase the number of enzyme molecules associated with hybridization and hence provides an increase in detectability; moreover, haptens (biotin, dinitrophenol, acetylaminofluorene analogues, digoxigenin, brominated or sulphonylated pyrimidines) used to label nucleic acid probes are not sensitive to elevated temperatures (42-80 degrees C), extended incubation times (several hours), detergents and organic solvents currently required in hybridization techniques. The application of the immunoenzymatic and related techniques to nucleic acid probing is reviewed, focussing on the strategies of non-radioactive hybridization, hapten-labelling of nucleic acids and methods for the immunodetection of the hybrids. PMID:1613257

Guesdon, J L

1992-06-24

400

Identification of Nucleic Acid Binding Sites on Translin-Associated Factor X (TRAX) Protein  

PubMed Central

Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity.

Gupta, Gagan Deep; Kumar, Vinay

2012-01-01

401

Identification of nucleic acid binding sites on translin-associated factor X (TRAX) protein.  

PubMed

Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity. PMID:22427937

Gupta, Gagan Deep; Kumar, Vinay

2012-01-01

402

The Effects of Borate Minerals on the Synthesis of Nucleic Acid Bases, Amino Acids and Biogenic Carboxylic Acids from Formamide  

NASA Astrophysics Data System (ADS)

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

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

2011-08-01

403

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.

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

2013-01-01

404

Peptide nucleic acid-mediated recombination for targeted genomic repair and modification.  

PubMed

The ability to directly manipulate the human genome to correct a disease-related mutation, introduce a sequence change that would lead to site-specific gene knockout, or increase gene expression is a very powerful tool with tremendous clinical value. Triplex formation by synthetic DNA-binding molecules such as peptide nucleic acids (PNAs) has been studied for over 20 years and much of the work in the last 10 years has shown its great promise in its use to direct site-specific gene modification for the use in gene therapy. In this chapter, detailed protocols are described for the design and use of triplex-forming PNAs to bind and mediate gene modification at specific chromosomal targets. Target site identification, PNA and donor oligonucleotide design, in vitro characterization of binding, optimization with reporter systems, as well as various methods to assess gene modification and isolate modified cells are described. PMID:24297362

Schleifman, Erica B; Glazer, Peter M

2014-01-01

405

Relevance of nucleic acid amplification techniques for diagnosis of respiratory tract infections in the clinical laboratory.  

PubMed Central

Clinical laboratories are increasingly receiving requests to perform nucleic acid amplification tests for the detection of a wide variety of infectious agents. In this paper, the efficiency of nucleic acid amplification techniques for the diagnosis of respiratory tract infections is reviewed. In general, these techniques should be applied only for the detection of microorganisms for which available diagnostic techniques are markedly insensitive or nonexistent or when turnaround times for existing tests (e.g., viral culture) are much longer than those expected with amplification. This is the case for rhinoviruses, coronaviruses, and hantaviruses causing a pulmonary syndrome, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Coxiella burnetii. For Legionella spp. and fungi, contamination originating from the environment is a limiting factor in interpretation of results, as is the difficulty in differentiating colonization and infection. Detection of these agents in urine or blood by amplification techniques remains to be evaluated. In the clinical setting, there is no need for molecular diagnostic tests for the diagnosis of Pneumocystis carinii. At present, amplification methods for Mycobacterium tuberculosis cannot replace the classical diagnostic techniques, due to their lack of sensitivity and the absence of specific internal controls for the detection of inhibitors of the reaction. Also, the results of interlaboratory comparisons are unsatisfactory. Furthermore, isolates are needed for susceptibility studies. Additional work remains to be done on sample preparation methods, comparison between different amplification methods, and analysis of results. The techniques can be useful for the rapid identification of M. tuberculosis in particular circumstances, as well as the rapid detection of most rifampin-resistant isolates. The introduction of diagnostic amplification techniques into a clinical laboratory implies a level of proficiency for excluding false-positive and false-negative results.

Ieven, M; Goossens, H

1997-01-01

406

Physical methods of nucleic acid transfer: general concepts and applications  

PubMed Central

Physical methods of gene (and/or drug) transfer need to combine two effects to deliver the therapeutic material into cells. The physical methods must induce reversible alterations in the plasma membrane to allow the direct passage of the molecules of interest into the cell cytosol. They must also bring the nucleic acids in contact with the permeabilized plasma membrane or facilitate access to the inside of the cell. These two effects can be achieved in one or more steps, depending upon the methods employed. In this review, we describe and compare several physical methods: biolistics, jet injection, hydrodynamic injection, ultrasound, magnetic field and electric pulse mediated gene transfer. We describe the physical mechanisms underlying these approaches and discuss the advantages and limitations of each approach as well as its potential application in research or in preclinical and clinical trials. We also provide conclusions, comparisons, and projections for future developments. While some of these methods are already in use in man, some are still under development or are used only within clinical trials for gene transfer. The possibilities offered by these methods are, however, not restricted to the transfer of genes and the complementary uses of these technologies are also discussed. As these methods of gene transfer may bypass some of the side effects linked to viral or biochemical approaches, they may find their place in specific clinical applications in the future. This article is part of a themed section on Vector Design and Drug Delivery. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009

Villemejane, Julien; Mir, Lluis M

2009-01-01

407

Intracellular fate of spherical nucleic acid nanoparticle conjugates.  

PubMed

Spherical nucleic acid (SNA) nanoparticle conjugates are a class of bionanomaterials that are extremely potent in many biomedical applications. Their unique ability to enter multiple mammalian cell types as single-entity agents arises from their novel three-dimensional architecture, which consists of a dense shell of highly oriented oligonucleotides chemically attached typically to a gold nanoparticle core. This architecture allows SNAs to engage certain cell surface receptors to facilitate entry. Here, we report studies aimed at determining the intracellular fate of SNAs and the trafficking events that occur inside C166 mouse endothelial cells after cellular entry. We show that SNAs traffic through the endocytic pathway into late endosomes and reside there for up to 24 h after incubation. Disassembly of oligonucleotides from the nanoparticle core is observed 16 h after cellular entry, most likely due to degradation by enzymes such as DNase II localized in late endosomes. Our observations point to these events being likely independent of core composition and treatment conditions, and they do not seem to be particularly dependent upon oligonucleotide sequence. Significantly and surprisingly, the SNAs do not enter the lysosomes under the conditions studied. To independently track the fate of the particle core and the fluorophore-labeled oligonucleotides that comprise its shell, we synthesized a novel class of quantum dot SNAs to determine that as the SNA structures are broken down over the 24 h time course of the experiment, the oligonucleotide fragments are recycled out of the cell while the nanoparticle core is not. This mechanistic insight points to the importance of designing and synthesizing next-generation SNAs that can bypass the degradation bottleneck imposed by their residency in late endosomes, and it also suggests that such structures might be extremely useful for endosomal signaling pathways by engaging receptors that are localized within the endosome. PMID:24841494

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

2014-05-28

408

Base pairing and base mis-pairing in nucleic acids  

NASA Technical Reports Server (NTRS)

In recent years we have learned that DNA is conformationally active. It can exist in a number of different stable conformations including both right-handed and left-handed forms. Using single crystal X-ray diffraction analysis we are able to discover not only additional conformations of the nucleic acids but also different types of hydrogen bonded base-base interactions. Although Watson-Crick base pairings are the predominant type of interaction in double helical DNA, they are not the only types. Recently, we have been able to examine mismatching of guanine-thymine base pairs in left-handed Z-DNA at atomic resolution (1A). A minimum amount of distortion of the sugar phosphate backbone is found in the G x T pairing in which the bases are held together by two hydrogen bonds in the wobble pairing interaction. Because of the high resolution of the analysis we can visualize water molecules which fill in to accommodate the other hydrogen bonding positions in the bases which are not used in the base-base interactions. Studies on other DNA oligomers have revealed that other types of non-Watson-Crick hydrogen bonding interactions can occur. In the structure of a DNA octamer with the sequence d(GCGTACGC) complexed to an antibiotic triostin A, it was found that the two central AT base pairs are held together by Hoogsteen rather than Watson-Crick base pairs. Similarly, the G x C base pairs at the ends are also Hoogsteen rather than Watson-Crick pairing. Hoogsteen base pairs make a modified helix which is distinct from the Watson-Crick double helix.

Wang, A. H. J.; Rich, A.

1986-01-01

409

Remote Enantioselection Transmitted by an Achiral Peptide Nucleic Acid Backbone  

NASA Technical Reports Server (NTRS)

short homochiral segment of DNA into a PNA helix could have guaranteed that the next short segment of DNA to be incorporated would have the same handedness as the first. Once two segments of the same handedness were present, the probability that a third segment would have the same handedness would increase, and so on. Evolution could then slowly dilute out the PNA part. This scenario would ultimately allow the formation of a chiral oligonucleotide by processes that are largely resistant to enantiomeric crossinhibition. It is important to note that the ligation of homochiral dinucleotides on a nucleic acid template would probably be at least as enantiospecific as the reaction that we have studied. The disadvantage of using chiral monomers as components of a replicating system arises from the difficulty of generating a first long homochiral template from a racemic mixture of monomers, although results of experiments designed to overcome this difficulty by employing homochiral tetramers have been reported.l l The probability of obtaining a homochiral n-mer from achiral substrates is approximately 1P-I if the nontemplate-directed extension of the primer is not enantioselective. Hence, it would be very hard to get started with a homochiral 40-mer, for example. No such difficulty exists in a scenario that originates with an achiral genetic material and in which the incorporation of very few chiral monomers in this achiral background gradually progresses towards homochirality. It seems possible that some PNA sequences could act as catalysts, analogous to ribozymes, even though PNA lacks clear metal binding sites. Although such catalysts could not be enantioselective, the incorporation of as few as two chiral nucleotides could then impose chiral specificity on the system. Furthermore, such patch chimeras could help to bridge the gap in catalytic potential between PNA and RNA, while guaranteeing enantioselectivity.

Kozlov, Igor A.; Orgel, Leslie E.; Nielsen, Peter E.

2000-01-01

410

Determination of nucleic acid by its enhancement effect on the fluorescence of Ellagic acid - Cationic surfactant system  

NASA Astrophysics Data System (ADS)

In this paper, nucleic acid can greatly enhance the fluorescence of Ellagic acid (EA) in the presence of cetylpyridine bromide (CPB). Experiments indicate that under the optimum conditions, the enhanced intensity of fluorescence is proportional to the concentration of nucleic acid in the range of 5.0 × 10 -9-3.5 × 10 -5 g mL -1 for hsDNA, 5.0 × 10 -9-3.5 × 10 -5 g mL -1 for ctDNA and 5.0 × 10 -9-3.5 × 10 -5 g mL -1 for yRNA. Their detection limits (S/N = 3) are 7.6 × 10 -9 g mL -1, 8.6 × 10 -9 g mL -1 and 6.1 × 10 -9 g mL -1, respectively. The method has been satisfactorily used for the determination of nucleic acid in actual samples. Resonance Light Scattering, Ultraviolet and other means are used to discuss its mechanism. It is considered that the charge-transfer complex EA-CPB aggregate in the extended nucleic acids by hydrogen bond and electric attraction. The hydrophobic microenvironment of nucleic acid makes the fluorescence intensity of EA-CPB-nucleic acid system much stronger.

Wang, Feng; Huang, Wei; Wang, Yanwei; Tang, Bo

2010-04-01

411

The Effects of Borate Minerals on the Synthesis of Nucleic Acid Bases, Amino Acids and Biogenic Carboxylic Acids from Formamide  

Microsoft Academic Search

The thermal condensation of formamide in the presence of mineral borates is reported. The products afforded are precursors\\u000a of nucleic acids, amino acids derivatives and carboxylic acids. The efficiency and the selectivity of the reaction was studied\\u000a in relation to the elemental composition of the 18 minerals analyzed. The possibility of synthesizing at the same time building\\u000a blocks of both

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

2011-01-01

412

Size-Dependent Infiltration and Optical Detection of Nucleic Acids in Nanoscale Pores  

Microsoft Academic Search

Experiments and complimentary simulations are presented to demonstrate the size-dependent infiltration and detection of variable length nucleic acids in porous silicon with controllable pore diameters in the range of 15-60 nm. The pore diameter must be tuned according to target molecule size in order to most effectively balance sensitivity and size-exclusion. A quantitative relationship between pore size (15-60 nm), nucleic

Jenifer L. Lawrie; Yang Jiao; Sharon M. Weiss

2010-01-01

413

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

NASA Technical Reports Server (NTRS)

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

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

1993-01-01

414

Determination of a universal nucleic acid extraction procedure for PCR detection of gastroenteritis viruses in faecal specimens  

Microsoft Academic Search

Four nucleic acid extraction protocols were examined for their suitability for extraction of the ssRNA, dsRNA and dsDNA genomes of gastroenteritis viruses, for PCR detection. Protocol (A), employed specimen lysis with guanidinium thiocyanate, extraction with phenol–chloroform–isoamyl alcohol and nucleic acid purification by size-fractionated silica particles. Protocol (B), utilised specimen lysis with guanidinium thiocyanate and nucleic acid purification by silica, followed

Nassar B. G Rasool; Stephan S Monroe; Roger I Glass

2002-01-01

415

Cationic Lipid-Nucleic Acid Complexes (Lipoplexes): from Physicochemical Properties to In Vitro and In Vivo Transfection Kits  

Microsoft Academic Search

\\u000a Lipoplexes are complexes formed spontaneously upon mixing of negatively charged nucleic acids (or other polyelectrolytes such\\u000a as proteins) with positively-charged lipid assemblies [1]( for definitions see [2]). The relevant nucleic acids include plasmid DNA (pDNA), linear DNA, single chain DNA, oligonucleotide (ODN), messenger\\u000a RNA, and silencing double stranded short RNA (siRNA) [1–3] The lipoplex-mediated nucleic acid delivery appears to be

Dmitri Simberg; Danielle Hirsch-Lerner; Nicolaas-Jan Zuidam; Simcha Even-Chen; Miryam Kerner; Hagit Eliyahu; Natalie Servel; Sarah Weisman; Alla Plis-Finarov; Yeshayahu Talmon; Yechezkel Barenholz

416

Comparison of nucleic acid content in populations of free-living and symbiotic Rhizobium meliloti by flow microfluorometry.  

PubMed Central

Populations of symbiotic Rhizobium meliloti extracted from alfalfa nodules were shown by flow microfluorometry to contain a significant number of bacteroids with higher nucleic acid content than the free-living rhizobia. Bacteroids with lower nucleic acid content than the free-living bacteria were not detected in significant quantities in these populations. These results indicate that the incapability of bacteroids to reestablish growth in nutrient media may not be caused by a decrease in nucleic acid content of the symbiotic rhizobia.

Paau, A S; Lee, D; Cowles, J R

1977-01-01

417

Resonance Rayleigh scattering study of the reaction of nucleic acids with thionine and its analytical application  

NASA Astrophysics Data System (ADS)

Resonance Rayleigh scattering (RRS) of the thionine (TH)-nucleic acids system and its analytical application have been studied. In pH 2.2 acidic buffer medium, some nucleic acids can react with TH to form TH-nucleic acids complex. This results in a great enhancement of RRS and the appearance of new RRS spectra. The RRS spectral characteristics of TH-ctDNA system, the affecting factors and the optimum conditions of the reaction have been investigated. The enhancement of the RRS signal is directly proportional to the concentration of nucleic acids in the range 0-10.0 ?g/ml for calf thymus DNA and 0-15.0 ?g/ml for yeast RNA, and its detection limits (3?) are 3.5 ng/ml for calf thymus DNA and 4.9 ng/ml for yeast RNA, respectively. The method shows a wide linear range and high sensitivity, and was applied to the determination of trace amounts of nucleic acid in synthetic samples and practical samples with satisfactory results. The bind properties for the interactions of TH with ctDNA were investigated using a Scatchard plot based on the measurement of the enhanced RRS data at 340 nm, and the binding number and intrinsic binding constant are 4.9 and 2.6×10 5 mol/dm 3, respectively.

Long, Xiufen; Bi, Shuping; Tao, Xiancong; Wang, Yongzhong; Zhao, Hong

2004-01-01

418

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

419

Nano-vectors for the Ocular Delivery of Nucleic Acid-based Therapeutics.  

PubMed

Nucleic acid-based therapeutics have gained a lot of interest for the treatment of diverse ophthalmic pathologies. The first to enter in clinic has been an oligonucleotide, Vitravene(®) for the treatment of cytomegalovirus infection. More recently, research on aptamers for the treatment of age related macular degeneration has led to the development of Macugen(®). Despite intense potential, effective ocular delivery of nucleic acids is a major challenge since therapeutic targets for nucleic acid-based drugs are mainly located in the posterior eye segment, requiring repeated invasive administration. Of late, nanotechnology-based nano-vectors have been developed in order to overcome the drawbacks of viral and other non-viral vectors. The diversity of nano-vectors allows for ease of use, flexibility in application, low-cost of production, higher transfection efficiency and enhanced genomic safety. Using nano-vector strategies, nucleic acids can be delivered either encapsulated or complexed with cationic lipids, polymers or peptides forming sustained release systems, which can be tailored according to the ocular tissue being targeted. The present review focuses on developments and advances in various nano-vectors for the ocular delivery of nucleic acid-based therapeutics, the barriers that such delivery systems face and methods to overcome them. PMID:21969738

Khar, R K; Jain, G K; Warsi, M H; Mallick, N; Akhter, S; Pathan, S A; Ahmad, F J

2010-11-01

420

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.

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

2014-01-01

421

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

PubMed Central

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

Shyur, Lie-Fen

2013-01-01

422

Shortening distance of forward and reverse primers for nucleic acid isothermal amplification.  

PubMed

Abstract Existent nucleic acid isothermal detection techniques for clinical diseases are difficult to promote greatly due to limitations in such aspects as methodology, costs of detection, amplification efficiency and conditions for operation. There is therefore an urgent need for a new isothermal amplification method with the characteristics of high accuracy, easy operation, short time of detection and low costs. We have devised a new method of nucleic acid isothermal amplification using Bst DNA polymerase under isothermal conditions (60-65°C). We call this method of amplification by shortening the distance between forward and reverse primers for nucleic acid isothermal amplification SDAMP. The results demonstrated that this technique is highly sensitive, specific and has short reaction times (40-60 min). Results of sequencing show that the products of SDAMP amplification are mainly polymers formed by series connection of monomers formed through linkage of forward primer and complementary sequences in reverse primer via a few bases. The method is different from current methods of nucleic acid amplification. Our study shows, however, that it is a specific method of nucleic acid isothermal amplification depending on interactions between primers and DNA template. PMID:24713573

Haitao, Qu; Wenchao, Zhang; Xiaohui, Zhang; Xiujun, Wang; Sulong, Li

2014-06-01

423

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

424

Recent advances in delivery of drug-nucleic acid combinations for cancer treatment.  

PubMed

Cancer treatment that uses a combination of approaches with the ability to affect multiple disease pathways has been proven highly effective in the treatment of many cancers. Combination therapy can include multiple chemotherapeutics or combinations of chemotherapeutics with other treatment modalities like surgery or radiation. However, despite the widespread clinical use of combination therapies, relatively little attention has been given to the potential of modern nanocarrier delivery methods, like liposomes, micelles, and nanoparticles, to enhance the efficacy of combination treatments. This lack of knowledge is particularly notable in the limited success of vectors for the delivery of combinations of nucleic acids with traditional small molecule drugs. The delivery of drug-nucleic acid combinations is particularly challenging due to differences in the physicochemical properties of the two types of agents. This review discusses recent advances in the development of delivery methods using combinations of small molecule drugs and nucleic acid therapeutics to treat cancer. This review primarily focuses on the rationale used for selecting appropriate drug-nucleic acid combinations as well as progress in the development of nanocarriers suitable for simultaneous delivery of drug-nucleic acid combinations. PMID:23624358

Li, Jing; Wang, Yan; Zhu, Yu; Oupický, David

2013-12-10

425

Synthetic nucleic acids delivered by exosomes: a potential therapeutic for generelated metabolic brain diseases.  

PubMed

Many brain diseases, including Alzheimer's disease, are associated with genetic abnormalities. The search for more effective therapeutic approaches involving nucleic acids like interfering RNA, antisense oligonucleotides and mRNA has drawn much attention in the development of alternatives to virus-based gene therapy. Potentially, nucleic acids could not only specifically down-regulate and degrade misfolded proteins, but also relieve protein deficiencies by directing the translation of functional proteins. However, clinical applications have been stalled by the lack of proper delivery systems. Exosomes are nano-scale extracellular vesicles secreted by nearly all somatic cells. Recent work has revealed that exosomes play special roles in intercellular communication via the horizontal transfer of various RNAs among cells. R