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‣ Tumor-targeted Chlorotoxin-coupled Nanoparticles for Nucleic Acid Delivery to Glioblastoma Cells: A Promising System for Glioblastoma Treatment

Costa, Pedro M.; Cardoso, Ana L.; Mendonça, Liliana S.; Serani, Angelo; Custódia, Carlos; Conceição, Mariana; Simões, Sérgio; Moreira, João N.; Almeida, Luis Pereira de; de Lima, M. C. P.
Fonte: The American Society of Gene & Cell Therapy Publicador: The American Society of Gene & Cell Therapy
Tipo: Artigo de Revista Científica
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The present work aimed at the development and application of a lipid-based nanocarrier for targeted delivery of nucleic acids to glioblastoma (GBM). For this purpose, chlorotoxin (CTX), a peptide reported to bind selectively to glioma cells while showing no affinity for non-neoplastic cells, was covalently coupled to liposomes encapsulating antisense oligonucleotides (asOs) or small interfering RNAs (siRNAs). The resulting targeted nanoparticles, designated CTX-coupled stable nucleic acid lipid particles (SNALPs), exhibited excellent features for in vivo application, namely small size (<180 nm) and neutral surface charge. Cellular association and internalization studies revealed that attachment of CTX onto the liposomal surface enhanced particle internalization into glioma cells, whereas no significant internalization was observed in noncancer cells. Moreover, nanoparticle-mediated miR-21 silencing in U87 human GBM and GL261 mouse glioma cells resulted in increased levels of the tumor suppressors PTEN and PDCD4, caspase 3/7 activation and decreased tumor cell proliferation. Preliminary in vivo studies revealed that CTX enhances particle internalization into established intracranial tumors. Overall, our results indicate that the developed targeted nanoparticles represent a valuable tool for targeted nucleic acid delivery to cancer cells. Combined with a drug-based therapy...

‣ Fractionation and identification of the nucleic acid-related substances secreted by Streptomyces aureofaciens

deCarvalho, A.; Malavolta, C. M.; Quirino, G. H.; Fernandes, JAD; Mengatti, J.; Contiero, Jonas; Oppermann, H. R.; Molinari, R.
Fonte: Editora Unesp Publicador: Editora Unesp
Tipo: Artigo de Revista Científica Formato: 71-87
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S. aureofaciens growth in a chemically defined medium was associated with the active secretion of nucleic acid-related substances in the medium. High secretion depended on low availability of phosphate, and fractionation showed 7 anionic substances were secreted as major components. When compared to 76 known purine and pyrimidine derivatives only erotic acid was identified. Cationic components are among the minor concentration components secreted which have been identified as cytosine, inosine, cytidine, adenine, guanine and, probably, 1-methyl-adenine.

‣ Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Lactobacillus spp. in milk samples

Machado, António; Almeida, Carina; Carvalho, Ana; Boyen, Filip; Haesebrouck, Freddy; Rodrigues, L. R.; Cerca, Nuno; Azevedo, N. F.
Fonte: Elsevier; Elsevier BV Publicador: Elsevier; Elsevier BV
Tipo: Artigo de Revista Científica
Publicado em //2013 Português
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Lactobacillus species constitute one of the dominant and beneficial bacteria in our body and are used in developed countries as a microbial adjuvant. Identification of these probiotic bacteria is traditionally performed by culture-based techniques. However, such methods are very time-consuming and can give inaccurate results, especially when Lactobacillus is present in mixed bacterial complex communities. Our study aimed to accurately identify Lactobacillus spp. using a novel Peptide Nucleic Acid (PNA) Fluorescence In Situ Hibridization (FISH) probe. The probe (Lac663) was tested on 36 strains belonging to different Lactobacillus species and on 20 strains of other bacterial species. The sensitivity and specificity of the method were 100% (95% confidence interval (CI), 88.0 to 100.0%) and 95.0% (95% CI, 73.1 to 99.7%), respectively. Additionally, we tested the applicability of the method on milk samples added with Lactobacillus strains at probiotic range concentrations and others taxonomically related bacteria, as well as pathogenic bacteria. The Lac663 probe bound exclusively to Lactobacillus strains and the described PNA-FISH method was capable of directly quantifying Lactobacillus spp. in concentrations at which these potential probiotic bacteria are considered to have an effective benefit on human health.

‣ Fluorescence in situ hybridization method using peptide nucleic acid probes for rapid detection of Lactobacillus and Gardnerella spp

Machado, António; Almeida, Carina; Salgueiro, D. A. L.; Henriques, Ana Filipa Frutuoso Mendes; Vaneechoutte, Mario; Haesebrouck, Freddy; Vieira, M. J.; Rodrigues, L. R.; Azevedo, N. F.; Cerca, Nuno
Fonte: BioMed Central Publicador: BioMed Central
Tipo: Artigo de Revista Científica
Publicado em //2013 Português
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Background: Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples. Results: Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora...

‣ Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method

Machado, António; Castro, J.; Cereija, Tatiana; Almeida, Carina; Cerca, Nuno
Fonte: PeerJ Inc Publicador: PeerJ Inc
Tipo: Artigo de Revista Científica
Publicado em 17/02/2015 Português
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Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situHybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.699.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis.

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

Chícharo, Alexandra; Chícharo, Luís
Fonte: Universidade do Algarve Publicador: Universidade do Algarve
Tipo: Artigo de Revista Científica
Publicado em //2008 Português
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Some of most used indicators in marine ecology are nucleic acid-derived indices. They can be divided by target levels in three groups: 1) at the organism level as ecophysiologic indicators, indicators such as RNA:DNA ratios, DNA:dry weight and RNA:protein, 2) at the population level, indicators such as growth rate, starvation incidence or fisheries impact indicators, and 3) at the community level, indicators such as trophic interactions, exergy indices and prey identification. The nucleic acids derived indices, especially RNA:DNA ratio, have been applied with success as indicators of nutritional condition, well been and growth in marine organisms. They are also useful as indicators of natural or anthropogenic impacts in marine population and communities, such as upwelling or dredge fisheries, respectively. They can help in understanding important issues of marine ecology such as trophic interactions in marine environment, fish and invertebrate recruitment failure and biodiversity changes, without laborious work of counting, measuring and identification of small marine organisms. Besides the objective of integrate nucleic acid derived indices across levels of organization, the paper will also include a general characterization of most used nucleic acid derived indices in marine ecology and also advantages and limitations of them. We can conclude that using indicators...

‣ A Computational Approach to Modeling Nucleic Acid Hairpin Structures

Tung, Chang-Shung
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1997 Português
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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.

‣ RNA Polymerase II with Open and Closed Trigger Loops: Active Site Dynamics and Nucleic Acid Translocation

Feig, Michael; Burton, Zachary F.
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em 20/10/2010 Português
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RNA polymerase II is the central eukaryotic enzyme in transcription from DNA to RNA. The dynamics of RNA polymerase II is described from molecular-dynamics simulations started from two crystal structures with open and closed trigger loop (TL) forms. Dynamic transitions between neutral and forward translocated states were observed, especially for the downstream DNA duplex. Dynamic rearrangements were also seen in the active site environment, including conformations in which the active site nucleotide assumed a possibly precatalytic conformation in close proximity to the terminal 3′-hydroxyl of the nascent RNA. Because nucleic acid translocation was observed primarily in the simulations with an open TL structure, whereas close approach of the active site nucleotide to the terminal RNA ribose predominantly occurred in the closed TL structure, a modified Brownian ratchet mechanism is proposed whereby thermally driven translocation is only possible with an open TL, and fidelity control and catalysis require TL closing.

‣ Polymerization and nucleic acid-binding properties of human L1 ORF1 protein

Callahan, Kathryn E.; Hickman, Alison B.; Jones, Charles E.; Ghirlando, Rodolfo; Furano, Anthony V.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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The L1 (LINE 1) retrotransposable element encodes two proteins, ORF1p and ORF2p. ORF2p is the L1 replicase, but the role of ORF1p is unknown. Mouse ORF1p, a coiled-coil-mediated trimer of ∼42-kDa monomers, binds nucleic acids and has nucleic acid chaperone activity. We purified human L1 ORF1p expressed in insect cells and made two findings that significantly advance our knowledge of the protein. First, in the absence of nucleic acids, the protein polymerizes under the very conditions (0.05 M NaCl) that are optimal for high (∼1 nM)-affinity nucleic acid binding. The non-coiled-coil C-terminal half mediates formation of the polymer, an active conformer that is instantly resolved to trimers, or multimers thereof, by nucleic acid. Second, the protein has a biphasic effect on mismatched double-stranded DNA, a proxy chaperone substrate. It protects the duplex from dissociation at 37°C before eventually melting it when largely polymeric. Therefore, polymerization of ORF1p seemingly affects its interaction with nucleic acids. Additionally, polymerization of ORF1p at its translation site could explain the heretofore-inexplicable phenomenon of cis preference—the favored retrotransposition of the actively translated L1 transcript, which is essential for L1 survival.

‣ Structure-Based Simulations of the Translocation Mechanism of the Hepatitis C Virus NS3 Helicase along Single-Stranded Nucleic Acid

Zheng, Wenjun; Tekpinar, Mustafa
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em 19/09/2012 Português
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The NS3 helicase of Hepatitis C virus is an ATP-fueled molecular motor that can translocate along single-stranded (ss) nucleic acid, and unwind double-stranded nucleic acids. It makes a promising antiviral target and an important prototype system for helicase research. Despite recent progress, the detailed mechanism of NS3 helicase remains unknown. In this study, we have combined coarse-grained (CG) and atomistic simulations to probe the translocation mechanism of NS3 helicase along ssDNA. At the residue level of detail, our CG simulations have captured functionally important interdomain motions of NS3 helicase and reproduced single-base translocation of NS3 helicase along ssDNA in the 3′–5′ direction, which is in good agreement with experimental data and the inchworm model. By combining the CG simulations with residue-specific perturbations to protein-DNA interactions, we have identified a number of key residues important to the translocation machinery that agree with previous structural and mutational studies. Additionally, our atomistic simulations with targeted molecular dynamics have corroborated the findings of CG simulations and further revealed key protein-DNA hydrogen bonds that break/form during the transitions. This study offers...

‣ The Nucleic Acid Database: new features and capabilities

Coimbatore Narayanan, Buvaneswari; Westbrook, John; Ghosh, Saheli; Petrov, Anton I.; Sweeney, Blake; Zirbel, Craig L.; Leontis, Neocles B.; Berman, Helen M.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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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.

‣ Dissecting the chemical interactions and substrate structural signatures governing RNA polymerase II trigger loop closure by synthetic nucleic acid analogues

Xu, Liang; Butler, Kyle Vincent; Chong, Jenny; Wengel, Jesper; Kool, Eric T.; Wang, Dong
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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The trigger loop (TL) of RNA polymerase II (Pol II) is a conserved structural motif that is crucial for Pol II catalytic activity and transcriptional fidelity. The TL remains in an inactive open conformation when the mismatched substrate is bound. In contrast, TL switches from an inactive open state to a closed active state to facilitate nucleotide addition upon the binding of the cognate substrate to the Pol II active site. However, a comprehensive understanding of the specific chemical interactions and substrate structural signatures that are essential to this TL conformational change remains elusive. Here we employed synthetic nucleotide analogues as ‘chemical mutation’ tools coupling with α-amanitin transcription inhibition assay to systematically dissect the key chemical interactions and structural signatures governing the substrate-coupled TL closure in Saccharomyces cerevisiae Pol II. This study reveals novel insights into understanding the molecular basis of TL conformational transition upon substrate binding during Pol II transcription. This synthetic chemical biology approach may be extended to understand the mechanisms of other RNA polymerases as well as other nucleic acid enzymes in future studies.

‣ The Nucleic Acid Database: new features and capabilities

Coimbatore Narayanan, Buvaneswari; Westbrook, John; Ghosh, Saheli; Petrov, Anton I.; Sweeney, Blake; Zirbel, Craig L.; Leontis, Neocles B.; Berman, Helen M.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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67.55274%
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.

‣ Simplified Sum-Over-States Approach for Predicting Resonance Raman Spectra. Application to Nucleic Acid Bases

Rappoport, Dmitrij; Shim, Sangwoo; Aspuru-Guzik, Alan
Fonte: American Chemical Society Publicador: American Chemical Society
Tipo: Artigo de Revista Científica
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Resonance Raman spectra provide a valuable probe into molecular excited-state structures and properties. Moreover, resonance enhancement is of importance for the chemical contribution to surface-enhanced Raman scattering. In this work, we introduce a simplified sum-over-states scheme for computing Raman spectra and Raman excitation profiles. The proposed sum-over-states approach uses derivatives of electronic excitation energies and transition dipole moments, which can be efficiently computed from time-dependent density functional theory. We analyze and interpret the resonance Raman spectra and Raman excitation profiles of nucleic acid bases using the present approach. Contributions of individual excited states under strictly resonant and non-resonant conditions are investigated, and smooth interpolation between both limiting cases is obtained.; Chemistry and Chemical Biology

‣ Validation of a fluorescence in situ hybridization method using peptide nucleic acid probes for detection of helicobacter pylori clarithromycin resistance in gastric biopsy specimens

Cerqueira, L.; Fernandes, RM.; Ferreira, RM.; Oleastro, M.; Carneiro, F.; Brandão, C.; Pimentel-Nunes, P.; Dinis-Ribeiro, M.; Figueiredo, C.; Keevil, CW.; Vieira, MJ.; Azevedo, NF.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em 17/04/2013 Português
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Here, we evaluated a previously established peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method as a new diagnostic test for Helicobacter pylori clarithromycin resistance detection in paraffin-embedded gastric biopsy specimens. Both a retrospective study and a prospective cohort study were conducted to evaluate the specificity and sensitivity of a PNA-FISH method to determine H. pylori clarithromycin resistance. In the retrospective study (n 30 patients), full agreement between PNA-FISH and PCR-sequencing was observed. Compared to the reference method (culture followed by Etest), the specificity and sensitivity of PNA-FISH were 90.9% (95% confidence interval [CI], 57.1% to 99.5%) and 84.2% (95% CI, 59.5% to 95.8%), respectively. In the prospective cohort (n 93 patients), 21 cases were positive by culture. For the patients harboring clarithromycin- resistant H. pylori, the method showed sensitivity of 80.0% (95% CI, 29.9% to 98.9%) and specificity of 93.8% (95% CI, 67.7% to 99.7%). These values likely represent underestimations, as some of the discrepant results corresponded to patients infected by more than one strain. PNA-FISH appears to be a simple, quick, and accurate method for detecting H. pylori clarithromycin resistance in paraffin-embedded biopsy specimens. It is also the only one of the methods assessed here that allows direct and specific visualization of this microorganism within the biopsy specimens...

‣ Polymeric micelles as versatile carriers for drugs and nucleic acids

El Sabahy, Mahmoud
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Thèse ou Mémoire numérique / Electronic Thesis or Dissertation
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Le cancer est la principale cause de mortalité au Canada. Les taxanes (e.g. le paclitaxel et le docétaxel (DCTX)) constituent des remèdes efficaces contre une série de tumeurs solides telles que les cancers du sein, du poumon et de l’ovaire. Par ailleurs, des acides nucléiques (e.g. les oligonucléotides antisens (AON) ou les petits ARN interférents (siRNAs)), capables de supprimer sélectivement certains oncogènes impliqués dans la carcinogénèse, sont actuellement étudiés pour traiter une large gamme de cancers. Bien que l’activité des taxanes et des acides nucléiques soit bien établie sur des modèles humains et/ou animaux, plusieurs aspects physico-chimiques et cliniques restent encore à améliorer. Leur solubilité limitée (pour les taxanes), leur dégradation rapide dans le sang (pour les acides nucléiques), leur élimination précoce, leur absence de sélectivité et leur toxicité envers les tissus sains sont les principaux facteurs limitant leur efficacité. C’est pourquoi de nombreux efforts ont porté sur l’élaboration de systèmes de vectorisation ciblés à base de polymères, dans le but de surmonter les problèmes associés aux thérapies actuelles. Dans cette thèse, deux types de micelles polymères ont été développés pour la vectorisation de DCTX et d’acides nucléiques. D’une part...

‣ The phosphate clamp: sequence selective nucleic acid binding profiles and conformational induction of endonuclease inhibition by cationic Triplatin complexes

Prisecaru, Andreea; Molphy, Zara; Kipping, Ralph G.; Peterson, Erica J.; Qu, Yun; Kellett, Andrew; Farrell, Nicholas P.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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The substitution-inert polynuclear platinum(II) complex (PPC) series, [{trans-Pt(NH3)2(NH2(CH2)nNH3)}2-μ-(trans-Pt(NH3)2(NH2(CH2)nNH2)2}](NO3)8, where n = 5 (AH78P), 6 (AH78 TriplatinNC) and 7 (AH78H), are potent non-covalent DNA binding agents where nucleic acid recognition is achieved through use of the ‘phosphate clamp' where the square-planar tetra-am(m)ine Pt(II) coordination units all form bidentate N–O–N complexes through hydrogen bonding with phosphate oxygens. The modular nature of PPC–DNA interactions results in high affinity for calf thymus DNA (Kapp ∼5 × 107 M−1). The phosphate clamp–DNA interactions result in condensation of superhelical and B-DNA, displacement of intercalated ethidium bromide and facilitate cooperative binding of Hoechst 33258 at the minor groove. The effect of linker chain length on DNA conformational changes was examined and the pentane-bridged complex, AH78P, was optimal for condensing DNA with results in the nanomolar region. Analysis of binding affinity and conformational changes for sequence-specific oligonucleotides by ITC, dialysis, ICP-MS, CD and 2D-1H NMR experiments indicate that two limiting modes of phosphate clamp binding can be distinguished through their conformational changes and strongly suggest that DNA condensation is driven by minor-groove spanning. Triplatin-DNA binding prevents endonuclease activity by type II restriction enzymes BamHI...

‣ Accelerated Sepsis Diagnosis by Seamless Integration of Nucleic Acid Purification and Detection

Hsu, Bang-Ning
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação
Publicado em //2014 Português
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Background The diagnosis of sepsis is challenging because the infection can be caused by more than 50 species of pathogens that might exist in the bloodstream in very low concentrations, e.g., less than 1 colony-forming unit/ml. As a result, among the current sepsis diagnostic methods there is an unsatisfactory trade-off between the assay time and the specificity of the derived diagnostic information. Although the present qPCR-based test is more specific than biomarker detection and faster than culturing, its 6 ~ 10 hr turnaround remains suboptimal relative to the 7.6%/hr rapid deterioration of the survival rate, and the 3 hr hands-on time is labor-intensive. To address these issues, this work aims to utilize the advances in microfluidic technologies to expedite and automate the ``nucleic acid purification - qPCR sequence detection'' workflow.

Methods and Results This task is evaluated to be best approached by combining immiscible phase filtration (IPF) and digital microfluidic droplet actuation (DM) on a fluidic device. In IPF, as nucleic acid-bound magnetic beads are transported from an aqueous phase to an immiscible phase, the carryover of aqueous contaminants is minimized by the high interfacial tension. Thus...

‣ Computational Molecular Engineering Nucleic Acid Binding Proteins and Enzymes

Reza, Faisal
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação Formato: 11061162 bytes; application/pdf
Publicado em //2010 Português
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Interactions between nucleic acid substrates and the proteins and enzymes that bind and catalyze them are ubiquitous and essential for reading, writing, replicating, repairing, and regulating the genomic code by the proteomic machinery. In this dissertation, computational molecular engineering furthered the elucidation of spatial-temporal interactions of natural nucleic acid binding proteins and enzymes and the creation of synthetic counterparts with structure-function interactions at predictive proficiency. We examined spatial-temporal interactions to study how natural proteins can process signals and substrates. The signals, propagated by spatial interactions between genes and proteins, can encode and decode information in the temporal domain. Natural proteins evolved through facilitating signaling, limiting crosstalk, and overcoming noise locally and globally. Findings indicate that fidelity and speed of frequency signal transmission in cellular noise was coordinated by a critical frequency, beyond which interactions may degrade or fail. The substrates, bound to their corresponding proteins, present structural information that is precisely recognized and acted upon in the spatial domain. Natural proteins evolved by coordinating substrate features with their own. Findings highlight the importance of accurate structural modeling. We explored structure-function interactions to study how synthetic proteins can complex with substrates. These complexes...

‣ Nanomechanics of Nucleic Acid Structures Investigated with AFM Based Force Spectroscopy

Rabbi, Mahir Haroon
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação
Publicado em //2010 Português
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Nucleic acids are subjected to many different mechanical loadings inside. These loadings could cause large deformations and conformational changes to these molecules. This is why the mechanical properties of nucleic acids are so important to their functions. Here we use a newly designed and built high-performance AFM force spectrometer, supplemented with molecular dynamics simulations and NMR spectroscopy to investigate the relationship between mechanical properties and structure of different nucleic acids.

To test the mechanical properties of nucleic acids, we successfully designed and purpose-built a single molecule puller, an instrument to physically stretch single molecules, at a fraction of the cost of a commercial AFM instrument. This instrument has similar force noise to hybrid instruments, while also exhibiting significantly lower drift, on the order of five times lower. This instrument allows the measurement of subtle transitions as a molecule is stretched. With the addition of a lock-in amplifier, we possibly could obtain better force resolution, the order of femtonewtons.

We find that helical structure does indeed have an effect on the mechanical properties of double-stranded DNA. As the A-form double helix has a shorter...