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Sample records for aptamer sequence structure

  1. Massively Parallel Interrogation of Aptamer Sequence, Structure and Function

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, N O; Tok, J B; Tarasow, T M

    2008-02-08

    Optimization of high affinity reagents is a significant bottleneck in medicine and the life sciences. The ability to synthetically create thousands of permutations of a lead high-affinity reagent and survey the properties of individual permutations in parallel could potentially relieve this bottleneck. Aptamers are single stranded oligonucleotides affinity reagents isolated by in vitro selection processes and as a class have been shown to bind a wide variety of target molecules. Methodology/Principal Findings. High density DNA microarray technology was used to synthesize, in situ, arrays of approximately 3,900 aptamer sequence permutations in triplicate. These sequences were interrogated on-chip for their ability to bind the fluorescently-labeled cognate target, immunoglobulin E, resulting in the parallel execution of thousands of experiments. Fluorescence intensity at each array feature was well resolved and shown to be a function of the sequence present. The data demonstrated high intra- and interchip correlation between the same features as well as among the sequence triplicates within a single array. Consistent with aptamer mediated IgE binding, fluorescence intensity correlated strongly with specific aptamer sequences and the concentration of IgE applied to the array. The massively parallel sequence-function analyses provided by this approach confirmed the importance of a consensus sequence found in all 21 of the original IgE aptamer sequences and support a common stem:loop structure as being the secondary structure underlying IgE binding. The microarray application, data and results presented illustrate an efficient, high information content approach to optimizing aptamer function. It also provides a foundation from which to better understand and manipulate this important class of high affinity biomolecules.

  2. Massively parallel interrogation of aptamer sequence, structure and function.

    Directory of Open Access Journals (Sweden)

    Nicholas O Fischer

    Full Text Available BACKGROUND: Optimization of high affinity reagents is a significant bottleneck in medicine and the life sciences. The ability to synthetically create thousands of permutations of a lead high-affinity reagent and survey the properties of individual permutations in parallel could potentially relieve this bottleneck. Aptamers are single stranded oligonucleotides affinity reagents isolated by in vitro selection processes and as a class have been shown to bind a wide variety of target molecules. METHODOLOGY/PRINCIPAL FINDINGS: High density DNA microarray technology was used to synthesize, in situ, arrays of approximately 3,900 aptamer sequence permutations in triplicate. These sequences were interrogated on-chip for their ability to bind the fluorescently-labeled cognate target, immunoglobulin E, resulting in the parallel execution of thousands of experiments. Fluorescence intensity at each array feature was well resolved and shown to be a function of the sequence present. The data demonstrated high intra- and inter-chip correlation between the same features as well as among the sequence triplicates within a single array. Consistent with aptamer mediated IgE binding, fluorescence intensity correlated strongly with specific aptamer sequences and the concentration of IgE applied to the array. CONCLUSION AND SIGNIFICANCE: The massively parallel sequence-function analyses provided by this approach confirmed the importance of a consensus sequence found in all 21 of the original IgE aptamer sequences and support a common stem:loop structure as being the secondary structure underlying IgE binding. The microarray application, data and results presented illustrate an efficient, high information content approach to optimizing aptamer function. It also provides a foundation from which to better understand and manipulate this important class of high affinity biomolecules.

  3. Screening and Identification of DNA Aptamers to Tyramine Using in Vitro Selection and High-Throughput Sequencing.

    Science.gov (United States)

    Valenzano, Stefania; De Girolamo, Annalisa; DeRosa, Maria C; McKeague, Maureen; Schena, Roberto; Catucci, Lucia; Pascale, Michelangelo

    2016-06-13

    Aptamers are synthetic single-stranded DNA or RNA sequences that can fold into tertiary structures allowing them to interact with and bind to targets with high affinity and specificity. This paper describes the first selection and identification of DNA aptamers able to recognize the biogenic amine tyramine. To successfully isolate aptamers to this challenging small molecule target, the SELEX methodology was adapted by combining a systematic strategy to increase the selection stringency and monitor enrichment success. As the benefits of applying high-throughput sequencing (HTS) in SELEX experiments is becoming more clear, this method was employed in combination with bioinformatics analysis to evaluate the utility of the selection strategy and to uncover new potential high affinity sequences. On the basis of the presence of consensus regions (sequence families) and family similarities (clusters), 15 putative aptamers to tyramine were identified. A recently described workflow approach to perform a primary screening and characterization of the aptamer candidates by microequilibrium dialysis and by microscale thermophoresis was next leveraged. These candidate aptamers exhibited dissociation constant (Kd) values in the range of 0.2-152 μM with aptamer Tyr_10 as the most promising one followed by aptamer Tyr_14. These aptamers could be used as promising molecular recognition tools for the development of inexpensive, robust and innovative biosensor platforms for the detection of tyramine in food and beverages.

  4. Structure analysis of free and bound states of an RNA aptamer against ribosomal protein S8 from Bacillus anthracis.

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    Davlieva, Milya; Donarski, James; Wang, Jiachen; Shamoo, Yousif; Nikonowicz, Edward P

    2014-01-01

    Several protein-targeted RNA aptamers have been identified for a variety of applications and although the affinities of numerous protein-aptamer complexes have been determined, the structural details of these complexes have not been widely explored. We examined the structural accommodation of an RNA aptamer that binds bacterial r-protein S8. The core of the primary binding site for S8 on helix 21 of 16S rRNA contains a pair of conserved base triples that mold the sugar-phosphate backbone to S8. The aptamer, which does not contain the conserved sequence motif, is specific for the rRNA binding site of S8. The protein-free RNA aptamer adopts a helical structure with multiple non-canonical base pairs. Surprisingly, binding of S8 leads to a dramatic change in the RNA conformation that restores the signature S8 recognition fold through a novel combination of nucleobase interactions. Nucleotides within the non-canonical core rearrange to create a G-(G-C) triple and a U-(A-U)-U quartet. Although native-like S8-RNA interactions are present in the aptamer-S8 complex, the topology of the aptamer RNA differs from that of the helix 21-S8 complex. This is the first example of an RNA aptamer that adopts substantially different secondary structures in the free and protein-bound states and highlights the remarkable plasticity of RNA secondary structure.

  5. Fluorescence-based strategies to investigate the structure and dynamics of aptamer-ligand complexes

    Science.gov (United States)

    Perez-Gonzalez, Cibran; Lafontaine, Daniel; Penedo, J.

    2016-08-01

    In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labelling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labelled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these

  6. Aptaligner: automated software for aligning pseudorandom DNA X-aptamers from next-generation sequencing data.

    Science.gov (United States)

    Lu, Emily; Elizondo-Riojas, Miguel-Angel; Chang, Jeffrey T; Volk, David E

    2014-06-10

    Next-generation sequencing results from bead-based aptamer libraries have demonstrated that traditional DNA/RNA alignment software is insufficient. This is particularly true for X-aptamers containing specialty bases (W, X, Y, Z, ...) that are identified by special encoding. Thus, we sought an automated program that uses the inherent design scheme of bead-based X-aptamers to create a hypothetical reference library and Markov modeling techniques to provide improved alignments. Aptaligner provides this feature as well as length error and noise level cutoff features, is parallelized to run on multiple central processing units (cores), and sorts sequences from a single chip into projects and subprojects.

  7. Reagentless, Structure-Switching, Electrochemical Aptamer-Based Sensors.

    Science.gov (United States)

    Schoukroun-Barnes, Lauren R; Macazo, Florika C; Gutierrez, Brenda; Lottermoser, Justine; Liu, Juan; White, Ryan J

    2016-06-12

    The development of structure-switching, electrochemical, aptamer-based sensors over the past ∼10 years has led to a variety of reagentless sensors capable of analytical detection in a range of sample matrices. The crux of this methodology is the coupling of target-induced conformation changes of a redox-labeled aptamer with electrochemical detection of the resulting altered charge transfer rate between the redox molecule and electrode surface. Using aptamer recognition expands the highly sensitive detection ability of electrochemistry to a range of previously inaccessible analytes. In this review, we focus on the methods of sensor fabrication and how sensor signaling is affected by fabrication parameters. We then discuss recent studies addressing the fundamentals of sensor signaling as well as quantitative characterization of the analytical performance of electrochemical aptamer-based sensors. Although the limits of detection of reported electrochemical aptamer-based sensors do not often reach that of gold-standard methods such as enzyme-linked immunosorbent assays, the operational convenience of the sensor platform enables exciting analytical applications that we address. Using illustrative examples, we highlight recent advances in the field that impact important areas of analytical chemistry. Finally, we discuss the challenges and prospects for this class of sensors.

  8. Reagentless, Structure-Switching, Electrochemical Aptamer-Based Sensors

    Science.gov (United States)

    Schoukroun-Barnes, Lauren R.; Macazo, Florika C.; Gutierrez, Brenda; Lottermoser, Justine; Liu, Juan; White, Ryan J.

    2016-06-01

    The development of structure-switching, electrochemical, aptamer-based sensors over the past ˜10 years has led to a variety of reagentless sensors capable of analytical detection in a range of sample matrices. The crux of this methodology is the coupling of target-induced conformation changes of a redox-labeled aptamer with electrochemical detection of the resulting altered charge transfer rate between the redox molecule and electrode surface. Using aptamer recognition expands the highly sensitive detection ability of electrochemistry to a range of previously inaccessible analytes. In this review, we focus on the methods of sensor fabrication and how sensor signaling is affected by fabrication parameters. We then discuss recent studies addressing the fundamentals of sensor signaling as well as quantitative characterization of the analytical performance of electrochemical aptamer-based sensors. Although the limits of detection of reported electrochemical aptamer-based sensors do not often reach that of gold-standard methods such as enzyme-linked immunosorbent assays, the operational convenience of the sensor platform enables exciting analytical applications that we address. Using illustrative examples, we highlight recent advances in the field that impact important areas of analytical chemistry. Finally, we discuss the challenges and prospects for this class of sensors.

  9. Sequence-dependent folding landscapes of adenine riboswitch aptamers

    Science.gov (United States)

    Lin, Jong-Chin; Hyeon, Changbong; Thirumalai, D.

    Prediction of the functions of riboswitches requires a quantitative description of the folding landscape so that the barriers and time scales for the conformational change in the switching region in the aptamer can be estimated. Using a combination of all atom molecular dynamics and coarse-grained model simulations we studied the response of adenine (A) binding add and pbuE A-riboswitches to mechanical force. The two riboswitches contain a structurally similar three-way junction formed by three paired helices, P1, P2, and P3, but carry out different functions. Using pulling simulations, with structures generated in MD simulations, we show that after P1 rips the dominant unfolding pathway in add A-riboswitch is the rupture of P2 followed by unraveling of P3. In the pbuE A-riboswitch, after P1 unfolds P3 ruptures ahead of P2. The order of unfolding of the helices, which is in accord with single molecule pulling experiments, is determined by the relative stabilities of the individual helices. Our results show that the stability of isolated helices determines the order of assembly and response to force in these non-coding regions. We use the simulated free energy profile for pbuE A-riboswitch to estimate the time scale for allosteric switching, which shows that this riboswitch is under kinetic control lending additional support to the conclusion based on single molecule pulling experiments. A consequence of the stability hypothesis is that a single point mutation (U28C) in the P2 helix of the add A-riboswitch, which increases the stability of P2, would make the folding landscapes of the two riboswitches similar. This prediction can be tested in single molecule pulling experiments.

  10. Comparison of the 'chemical' and 'structural' approaches to the optimization of the thrombin-binding aptamer.

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    Olga Tatarinova

    Full Text Available Noncanonically structured DNA aptamers to thrombin were examined. Two different approaches were used to improve stability, binding affinity and biological activity of a known thrombin-binding aptamer. These approaches are chemical modification and the addition of a duplex module to the aptamer core structure. Several chemically modified aptamers and the duplex-bearing ones were all studied under the same conditions by a set of widely known and some relatively new methods. A number of the thrombin-binding aptamer analogs have demonstrated improved characteristics. Most importantly, the study allowed us to compare directly the two approaches to aptamer optimization and to analyze their relative advantages and disadvantages as well as their potential in drug design and fundamental studies.

  11. Acyclic identification of aptamers for human alpha-thrombin using over-represented libraries and deep sequencing.

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    Gillian V Kupakuwana

    Full Text Available BACKGROUND: Aptamers are oligonucleotides that bind proteins and other targets with high affinity and selectivity. Twenty years ago elements of natural selection were adapted to in vitro selection in order to distinguish aptamers among randomized sequence libraries. The primary bottleneck in traditional aptamer discovery is multiple cycles of in vitro evolution. METHODOLOGY/PRINCIPAL FINDINGS: We show that over-representation of sequences in aptamer libraries and deep sequencing enables acyclic identification of aptamers. We demonstrated this by isolating a known family of aptamers for human α-thrombin. Aptamers were found within a library containing an average of 56,000 copies of each possible randomized 15mer segment. The high affinity sequences were counted many times above the background in 2-6 million reads. Clustering analysis of sequences with more than 10 counts distinguished two sequence motifs with candidates at high abundance. Motif I contained the previously observed consensus 15mer, Thb1 (46,000 counts, and related variants with mostly G/T substitutions; secondary analysis showed that affinity for thrombin correlated with abundance (K(d = 12 nM for Thb1. The signal-to-noise ratio for this experiment was roughly 10,000∶1 for Thb1. Motif II was unrelated to Thb1 with the leading candidate (29,000 counts being a novel aptamer against hexose sugars in the storage and elution buffers for Concanavilin A (K(d = 0.5 µM for α-methyl-mannoside; ConA was used to immobilize α-thrombin. CONCLUSIONS/SIGNIFICANCE: Over-representation together with deep sequencing can dramatically shorten the discovery process, distinguish aptamers having a wide range of affinity for the target, allow an exhaustive search of the sequence space within a simplified library, reduce the quantity of the target required, eliminate cycling artifacts, and should allow multiplexing of sequencing experiments and targets.

  12. Rational design of a structure-switching DNA aptamer for potassium ions

    Science.gov (United States)

    Catherine, Andrew T.; Shishido, Stephanie N.; Robbins-Welty, Gregg A.; Diegelman-Parente, Amy

    2014-01-01

    Structure-switching molecules provide a unique means for analyte detection, generating a response to analyte concentration through a binding-specific conformational change between non-binding and binding-competent states. While most ligand-binding molecules are not structure switching by default, many can be engineered to be so through the introduction of an alternative non-binding (and thus non-signalling) conformation. This population-shift mechanism is particularly effective with oligonucleotides and has led to the creation of structure-switching aptamers for many target ligands. Here, we report the rational design of structure-switching DNA aptamers, based on the thrombin binding aptamer (TBA), that bind potassium with affinities that bridge the gap between previously reported weak-binding and strong-binding aptamers. We also demonstrate a correlation between the free energy of the experimentally determined binding affinity for potassium and the computationally estimated free energy of the alternative (non-binding) structure. PMID:25352996

  13. Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode

    Energy Technology Data Exchange (ETDEWEB)

    Liu Xianggang [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Cheng Ziqiang, E-mail: czqsd@126.com [College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, Shandong (China); Fan Hai [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Ai Shiyun, E-mail: ashy@sdau.edu.cn [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Han Ruixia [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China)

    2011-07-15

    Highlights: > A sensitive electrochemical biosensor for the detection of gene sequence was developed. > The biosensor was assembled by MWNT, polypyrrole nanowires and gold nanoparticles. > The hybrid nanomaterials could provide a porous structure with good properties. > The biosensor has highly selectivity and sensitivity. > The design strategy is expected to have extensive applications in other biosensors - Abstract: A sensitive electrochemical method for the detection of avian influenza virus (AIV) H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode was developed. To enhance the selectivity and sensitivity, the modified electrode was assembled with multi-wall carbon nanotubes (MWNT), polypyrrole nanowires (PPNWs) and gold nanoparticles (GNPs). This electrode offered a porous structure with a large effective surface area, highly electrocatalytic activities and electronic conductivity. Therefore, the amount of DNA aptamer immobilized onto the electrode was increased while the accessibility of the detection target was maintained. The biosensor is based on the hybridization and preferred orientation of a DNA aptamer immobilized onto a modified electrode surface with its target (H5N1 specific sequence) present in solution. It is selective for the H5N1 specific sequence, and the signal of the indicator was approximately linear to log(concentration) of the H5N1 specific sequence from 5.0 x 10{sup -12} to 1.0 x 10{sup -9} M (R = 0.9863) with a detection limit of 4.3 x 10{sup -13} M. These studies showed that the new hybrid nanomaterial (MWNT/PPNWs/GNPs) and the DNA aptamer could be used to fabricate an electrochemical biosensor for gene sequence detection. Furthermore, this design strategy is expected to have extensive applications in other biosensors.

  14. Programmed dissociation of dimer and trimer origami structures by aptamer-ligand complexes.

    Science.gov (United States)

    Wu, Na; Willner, Itamar

    2017-01-26

    Dimer- and trimer-origami frames are bridged by duplexes that include caged, sequence-specific, anti-ATP and/or anti-cocaine aptamer sequences. The programmed dissociation of the origami dimers or trimers in the presence of ATP and/or cocaine ligands is demonstrated. The processes are followed by AFM imaging and by electrophoretic experiments.

  15. Structure-affinity relationship of the cocaine-binding aptamer with quinine derivatives.

    Science.gov (United States)

    Slavkovic, Sladjana; Altunisik, Merve; Reinstein, Oren; Johnson, Philip E

    2015-05-15

    In addition to binding its target molecule, cocaine, the cocaine-binding aptamer tightly binds the alkaloid quinine. In order to understand better how the cocaine-binding aptamer interacts with quinine we have used isothermal titration calorimetry-based binding experiments to study the interaction of the cocaine-binding aptamer to a series of structural analogs of quinine. As a basis for comparison we also investigated the binding of the cocaine-binding aptamer to a set of cocaine metabolites. The bicyclic aromatic ring on quinine is essential for tight affinity by the cocaine-binding aptamer with 6-methoxyquinoline alone being sufficient for tight binding while the aliphatic portion of quinine, quinuclidine, does not show detectable binding. Compounds with three fused aromatic rings are not bound by the aptamer. Having a methoxy group at the 6-position of the bicyclic ring is important for binding as substituting it with a hydrogen, an alcohol or an amino group all result in lower binding affinity. For all ligands that bind, association is driven by a negative enthalpy compensated by unfavorable binding entropy.

  16. Development of radiopharmaceuticals based on aptamers: selection and characterization of DNA aptamers for CEA

    Energy Technology Data Exchange (ETDEWEB)

    Correa, C.R.; Andrade, A.S.R., E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil); Augusto-Pinto, L. [BioAptus, Belo Horizonte, MG (Brazil); Goes, A.M., E-mail: goes@icb.ufmg.br [Departamento de Imunologia e Bioquimica. Instituto de Ciencias Biologicas. Universidade Federal de Minas Gerais. Belo Horizonte, MG (Brazil)

    2011-07-01

    Colorectal cancer is among the top four causes of cancer deaths worldwide. Carcinoembryonic antigen (CEA) is a complex intracellular glycoprotein produced by about 90% of colorectal cancers. CEA has been identified as an attractive target for cancer research because of its pattern of expression in the surface cell and its likely functional role in tumorigenesis. Research on the rapid selection of ligands based on the SELEX (systematic evolution of ligands by exponential enrichment) forms the basis for the development of high affinity and high specificity molecules, which can bind to surface determinants of tumour cells, like CEA. The oligonucleotides ligands generated in this technique are called aptamers. Aptamers can potentially find applications as therapeutic or diagnostic tools for many kind of diseases, like a tumor. Aptamers offer low immunogenicity, good tumour penetration, rapid uptake and fast systemic clearance, which favour their application as effective vehicles for radiotherapy. In addition aptamers can be labeled with different radioactive isotopes. The aim of this work was select aptamers binding to the CEA tumor marker. The aptamers are obtained through by SELEX, in which aptamers are selected from a library of random sequences of synthetic DNA by repetitive binding of the oligonucleotides to target molecule (CEA). Analyses of the secondary structure of the aptamers were determined using the m fold toll. Three aptamers were selected to binding assay with target cells. These aptamers were confirmed to have affinity and specific binding for T84 cell line (target cell), showed by confocal imaging. We are currently studying the potential efficacy of these aptamers as targeted radiopharmaceuticals, for use as imaging agents or therapeutic applications. The development of aptamers specific to CEA open new perspectives for colorectal cancer diagnosis and treatment. Acknowledgments: This investigation was supported by the Centro de Desenvolvimento da

  17. Selection of DNA Aptamers for Ovarian Cancer Biomarker CA125 Using One-Pot SELEX and High-Throughput Sequencing

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    Delia J. Scoville

    2017-01-01

    Full Text Available CA125 is a mucin glycoprotein whose concentration in serum correlates with a woman’s risk of developing ovarian cancer and also indicates response to therapy in diagnosed patients. Accurate detection of this large, complex protein in patient samples is of great clinical relevance. We suggest that powerful new diagnostic tools may be enabled by the development of nucleic acid aptamers with affinity for CA125. Here, we report on our use of One-Pot SELEX to isolate single-stranded DNA aptamers with affinity for CA125, followed by high-throughput sequencing of the selected oligonucleotides. This data-rich approach, combined with bioinformatics tools, enabled the entire selection process to be characterized. Using fluorescence anisotropy and affinity probe capillary electrophoresis, the binding affinities of four aptamer candidates were evaluated. Two aptamers, CA125_1 and CA125_12, both without primers, were found to bind to clinically relevant concentrations of the protein target. Binding was differently influenced by the presence of Mg2+ ions, being required for binding of CA125_1 and abrogating binding of CA125_12. In conclusion, One-Pot SELEX was found to be a promising selection method that yielded DNA aptamers to a clinically important protein target.

  18. In vitro Selection of DNA Aptamers and Fluorescence-Based Recognition for Rapid Detection Listeria monocytogenes

    Institute of Scientific and Technical Information of China (English)

    LIU Guo-qing; LIAN Ying-qi; GAO Chao; YU Xiao-feng; ZHU Ming; ZONG Kai; CHEN Xue-jiao; YAN Yi

    2014-01-01

    Aptamers are speciifc nucleic acid sequences that can bind to a wide range of nucleic acid and non-nucleic acid targets with high afifnity and speciifcity. Nucleic acid aptamers are selected in vitro from single stranded DNA or RNA ligands containing random sequences of up to a few hundred nucleotides. Systematic evolution of ligands by exponential enrichment (SELEX) was used to select and PCR amplify DNA sequences (aptamers) capable of binding to and detecting Listeria monocytogenes, one of the major food-borne pathogens. A simpliifed afifnity separation approach was employed, in which L. monocytogenes in exponential (log) phase of growth was used as the separation target. A lfuorescently-labeled aptamer assay scheme was devised for detecting L. monocytogenes. This report described a novel approach to the detection of L. monocytogenes using DNA aptamers. Aptamers were developed by nine rounds of SELEX. A high afifnity aptamer was successfully selected from the initial random DNA pool, and its secondary structure was also investigated. One of aptamers named e01 with the highest afifnity was further tested in aptamer-peroxidase and aptamer-lfuorescence staining protocols. This study has proved the principle that the whole-cell SELEX could be a promising technique to design aptamer-based molecular probes for dectection of pathogenic microorganisms without tedious isolation and puriifcation of complex markers or targets.

  19. A colorimetric detection method of pesticide acetamiprid by fine-tuning aptamer length.

    Science.gov (United States)

    Tian, Yu; Wang, Yuan; Sheng, Zhi; Li, Tingting; Li, Xu

    2016-11-15

    This work investigates the effect of shortening aptamer sequences on the colorimetric detection of acetamiprid using aptamer-wrapped gold nanoparticles (AuNPs). Truncated 37-mer and 25-mer aptamers were generated by deleting excess flanking nucleotides from parental 49-mer acetamiprid-target aptamer. In comparing the responses of the three sequences, truncated aptamers did not improve the ability to discriminate against other tested pesticides. However, comparison between 49-mer and other shorter aptamers showed that shortening aptamer sequences through removing excess flanking nucleotides outsides of binding region improved colorimetric sensitivity for acetamiprid by 3.3 fold. Due to excess bases, the target-bound aptamer might still adhere to AuNPs, resulting in incomplete dissociation of aptamer from AuNPs and therefore the suppression of aggregation responses. This work provides further insight to the effects of aptamer structure on detection of the target, as well as a method by fine-tuning aptamer length for rapid detection of pesticide residues in environments or food.

  20. Rational design of a structure-switching DNA aptamer for potassium ions

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    Andrew T. Catherine

    2014-01-01

    Full Text Available Structure-switching molecules provide a unique means for analyte detection, generating a response to analyte concentration through a binding-specific conformational change between non-binding and binding-competent states. While most ligand-binding molecules are not structure switching by default, many can be engineered to be so through the introduction of an alternative non-binding (and thus non-signalling conformation. This population-shift mechanism is particularly effective with oligonucleotides and has led to the creation of structure-switching aptamers for many target ligands. Here, we report the rational design of structure-switching DNA aptamers, based on the thrombin binding aptamer (TBA, that bind potassium with affinities that bridge the gap between previously reported weak-binding and strong-binding aptamers. We also demonstrate a correlation between the free energy of the experimentally determined binding affinity for potassium and the computationally estimated free energy of the alternative (non-binding structure.

  1. Targeting of Antibodies using Aptamers

    OpenAIRE

    2003-01-01

    The chapter presents a methodology for the rapid selection of aptamers against antibody targets. It is a detailed account of the various methodological steps that describe the selection of aptamers, including PCR steps, buffers to be used, target immobilisation, partitioning and amplification of aptamers, clonning and sequencing, to results in high affinity and specificity ligands for the chosen target antibody.

  2. Label-free fluorescent aptasensor for potassium ion using structure-switching aptamers and berberine

    Science.gov (United States)

    Guo, Yanqing; Chen, Yanxia; Wei, Yanli; Li, Huanhuan; Dong, Chuan

    2015-02-01

    A simple, rapid and label-free fluorescent aptasensor was fabricated for the detection of potassium ion (K+ ion) in aqueous solution using K+ ion-stabilized single stranded DNA (ssDNA) with G-rich sequence as the recognition element and a fluorescent dye, berberine, as the fluorescence probe. In the presence of K+ ion, the G-rich ssDNA is promoted to form the aptamer-target complex with a G-quadruplex conformation, and berberine binding to the G-quadruplex structure results in the enhancement of its fluorescence. The fluorescence intensity of the sensing system displayed a calibration response for K+ ion in the range of 0-1600 μM with a detection limit of 31 nM (S/N = 3) and a relative standard deviation (RSD) of 0.45%. This label-free fluorescence aptasensor is conveniently and effectively applicable for analysis of K+ ion in blood serum samples with the recovery range of 81.7-105.3%. The assay for detection of potassium ion is easy, economical, robust, and stable in rough conditions.

  3. Structural Dynamics of Thrombin-Binding DNA Aptamer d(GGTTGGTGTGGTTGG) Quadruplex DNA Studied by Large-Scale Explicit Solvent Simulations.

    Science.gov (United States)

    Reshetnikov, Roman; Golovin, Andrey; Spiridonova, Vera; Kopylov, Alexei; Šponer, Jiří

    2010-10-12

    The thrombin-binding aptamer (15-TBA) is a 15-mer DNA oligonucleotide with sequence d(GGTTGGTGTGGTTGG). 15-TBA folds into a quadruplex DNA (G-DNA) structure with two planar G-quartets connected by three single-stranded loops. The arrangement of the 15-TBA-thrombin complex is unclear, particularly with respect to the precise 15-TBA residues that interact with the thrombin structure. Our present understanding suggests either the 15-TBA single stranded loops containing sequential thymidines (TT) or alternatively a single-stranded loop, containing a guanine flanked by 2 thymidines (TGT), physically associates with thrombin protein. In the present study, the explicit solvent molecular dynamics (MD) simulation method was utilized to further analyze the 15-TBA-thrombin three-dimensional structure. Functional annotation of the loop residues was made with long simulations in the parmbsc0 force field. In total, the elapsed time of simulations carried out in this study exceeds 12 microseconds, substantially surpassing previous G-DNA simulation reports. Our simulations suggest that the TGT-loop function is to stabilize the structure of the aptamer, while the TT-loops participate in direct binding to thrombin. The findings of the present report advance our understanding of the molecular structure of the 15-TBA-thrombin structure further enabling the construction of biosensors for aptamer bases and the development of anticoagulant agents.

  4. In vitro evaluation of radiolabeled aptamers for colon carcinoma diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Correa, C.R.; Ferreira, I.M; Santos, S.R.; Faria, L.S.; Andrade, A.S.R., E-mail: crisrcorrea@gmail.com, E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil); Goes, A.M., E-mail: goes@icb.ufmg.br [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Inst. de Ciencias Biologicas. Dept. de Imunologia e Bioquimica

    2013-07-01

    Cancer is a leading cause of death worldwide, representing a major public health problem worldwide. Colorectal cancers accounts around 8% of all deaths for cancer in 2008, is the fourth most lethal. Many colorectal cancer markers, such as carcinoembryonic antigen (CEA), A33, and CSA-p, have been studied as the therapeutic targets in preclinical or clinical settings. CEA is a complex intracellular glycoprotein produced by about 90% of colorectal cancers. Since its discovery in 1965, a very large number of studies have been carried out to determine the effectiveness of CEA as clinically useful tumor markers. Aptamers are short single-stranded nucleic acid oligomers (DNA or RNA) that can form specific and complex three-dimensional structures which can bind with high affinity to specific targets, they are functionally equivalent of antibodies. Aptamers have the advantage of being highly specific, relatively small size, and non-immunogenic. The aim of this study was develop anti-CEA aptamers for use as imaging agents. The aptamers are obtained through by SELEX (systematic evolution of ligands by exponential enrichment), in which aptamers are selected from a library of random sequences of synthetic DNA by repetitive binding of the oligonucleotides to target molecule. These aptamers were confirmed to have affinity and specific binding for T84 cell line (target cell), showed by fluorescence microscopic images. Individual aptamers sequences that bound T84 cells were {sup 32}P-radiolabeled and incubated at different concentrations on cell monolayers, to monitor the aptamers affinity binding. The selected aptamers can identify colon cancer cell line. This aptamers could be further developed for early disease detection as radiopharmaceuticals, as well as prognostic markers, of colorectal cancers. (author)

  5. An aptamer beacon responsive to botulinum toxins.

    Science.gov (United States)

    Bruno, John G; Richarte, Alicia M; Carrillo, Maria P; Edge, Allison

    2012-01-15

    Sixty candidate DNA aptamers were developed against botulinum neurotoxin (BoNT) type A light chain (LC) from ten rounds of selection, resulting in several identical sequences. Secondary structures of the identical aptamers were compared to structures of previously reported BoNT A DNA aptamers. A series of ten candidate loop structures were selected from this comparison as potential binding pockets and aptamer beacons. These candidate beacons were synthesized with 5'-TYE 665 and 3'-Iowa Black quencher labels for comparison of fluorescence levels as a function of BoNT A LC concentration. Only three of the ten candidates exhibited any fluorescence response to increasing levels of BoNT A LC. However, of the two most responsive candidates, one represented a subset loop of the larger more intensely fluorescent double-looped structure, designated Beacon 10. This beacon yielded a lower limit of detection of 1 ng/mL in buffer using a spectrofluorometer and a portable handheld fluorometer, but also responded substantially to BoNT A, B, E holotoxins and heavy or light chain components even in a dilute soil suspension, but not in 50% human serum. Beacon 10 did not respond strongly to a variety of other divergent peptides, suggesting that it is relatively specific to the level of botulinum toxins and is only useful for environmental testing. Beacon 10 also shared short sequence segments with other published BoNT aptamer DNA sequences, suggesting that these may be points of physical contact between the aptamers and BoNTs.

  6. Aptamer-Gated Nanoparticles for Smart Drug Delivery

    Directory of Open Access Journals (Sweden)

    Huseyin Avni Oktem

    2011-08-01

    Full Text Available Aptamers are functional nucleic acid sequences which can bind specific targets. An artificial combinatorial methodology can identify aptamer sequences for any target molecule, from ions to whole cells. Drug delivery systems seek to increase efficacy and reduce side-effects by concentrating the therapeutic agents at specific disease sites in the body. This is generally achieved by specific targeting of inactivated drug molecules. Aptamers which can bind to various cancer cell types selectively and with high affinity have been exploited in a variety of drug delivery systems for therapeutic purposes. Recent progress in selection of cell-specific aptamers has provided new opportunities in targeted drug delivery. Especially functionalization of nanoparticles with such aptamers has drawn major attention in the biosensor and biomedical areas. Moreover, nucleic acids are recognized as an attractive building materials in nanomachines because of their unique molecular recognition properties and structural features. A active controlled delivery of drugs once targeted to a disease site is a major research challenge. Stimuli-responsive gating is one way of achieving controlled release of nanoparticle cargoes. Recent reports incorporate the structural properties of aptamers in controlled release systems of drug delivering nanoparticles. In this review, the strategies for using functional nucleic acids in creating smart drug delivery devices will be explained. The main focus will be on aptamer-incorporated nanoparticle systems for drug delivery purposes in order to assess the future potential of aptamers in the therapeutic area. Special emphasis will be given to the very recent progress in controlled drug release based on molecular gating achieved with aptamers.

  7. Utilizing a Key Aptamer Structure-Switching Mechanism for the Ultrahigh Frequency Detection of Cocaine.

    Science.gov (United States)

    Neves, Miguel A D; Blaszykowski, Christophe; Thompson, Michael

    2016-03-15

    Aptasensing of small molecules remains a challenge as detection often requires the use of labels or signal amplification methodologies, resulting in both difficult-to-prepare sensor platforms and multistep, complex assays. Furthermore, many aptasensors rely on the binding mechanism or structural changes associated with target capture by the aptameric probe, resulting in a detection scheme customized to each aptamer. It is in this context that we report herein a sensitive cocaine aptasensor that offers both real-time and label-free measurement capabilities. Detection relies on the electromagnetic piezoelectric acoustic sensor (EMPAS) platform. The sensing interface consists of a S-(11-trichlorosilyl-undecanyl)benzenethiosulfonate (BTS) adlayer-coated quartz disc onto which a structure-switching cocaine aptamer (MN6) is immobilized, completing the preparation of the MN6 cocaine aptasensor (M6CA). The EMPAS system has recently been employed as the foundation of a cocaine aptasensor based on a structurally rigid cocaine aptamer variant (MN4), an aptasensor referred to by analogy as M4CA. M6CA represents a significant increase in terms of analytical performance, compared to not only M4CA but also other cocaine aptamer-based sensors that do not rely on signal amplification, producing an apparent K(d) of 27 ± 6 μM and a 0.3 μM detection limit. Remarkably, the latter is in the range of that achieved by cocaine aptasensors relying on signal amplification. Furthermore, M6CA proved to be capable not only of regaining its cocaine-binding ability via simple buffer flow over the sensing interface (i.e., without the necessity to implement an additional regeneration step, such as in the case of M4CA), but also of detecting cocaine in a multicomponent matrix possessing potentially assay-interfering species. Finally, through observation of the distinct shape of its response profiles to cocaine injection, demonstration was made that the EMPAS system in practice offers the

  8. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    Full Text Available A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

  9. DNA Microarrays for Aptamer Identification and Structural Characterization

    Science.gov (United States)

    2012-09-01

    serum albumin Cy3 Cyanine dye3 Cy5 Cyanine dye5 Cy3-BSA Cyanine dye3 conjugated to bovine serum albumin Cy3-Estradiol Cyanine dye3 conjugated to...estradiol antibody Cy3-IgE Cyanine dye3 conjugated to Immunoglobulin E Cy3-NPYsc Cyanine dye3 conjugated to scrambled neuropeptide Y sequence Cy3-SA... Cyanine dye3 conjugated to streptavidin Cy3-thrombin Cyanine dye3 conjugated to thrombin DNA deoxyribonucleic acid D/P dye-to-protein ratio

  10. DNA aptamer beacon assay for C-telopeptide and handheld fluorometer to monitor bone resorption.

    Science.gov (United States)

    Bruno, John Gordon; Carrillo, Maria P; Phillips, Taylor; Hanson, Douglas; Bohmann, Jonathan A

    2011-09-01

    A novel DNA aptamer beacon is described for quantification of a 26-amino acid C-telopeptide (CTx) of human type I bone collagen. One aptamer sequence and its reverse complement dominated the aptamer pool (31.6% of sequenced clones). Secondary structures of these aptamers were examined for potential binding pockets. Three-dimensional computer models which analyzed docking topologies and binding energies were in agreement with empirical fluorescence experiments used to select one candidate loop for beacon assay development. All loop structures from the aptamer finalists were end-labeled with TYE 665 and Iowa Black quencher for comparison of beacon fluorescence levels as a function of CTx concentration. The optimal beacon, designated CTx 2R-2h yielded a low ng/ml limit of detection using a commercially available handheld fluorometer. The CTx aptamer beacon bound full-length 26-amino acid CTx peptide, but not a shorter 8-amino acid segment of CTx peptide which is a common target for commercial CTx ELISA kits. The prototype assay was shown to detect CTx peptide from human urine after creatinine and urea were removed by size-exclusion chromatography to prevent nonspecific denaturing of the aptamer beacon. This work demonstrates the potential of aptamer beacons to be utilized for rapid and sensitive bone health monitoring in a handheld or point-of-care format.

  11. In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies.

    Science.gov (United States)

    Lipi, Farhana; Chen, Suxiang; Chakravarthy, Madhuri; Rakesh, Shilpa; Veedu, Rakesh N

    2016-12-01

    Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries.

  12. The Toolbox for Modified Aptamers.

    Science.gov (United States)

    Lapa, Sergey A; Chudinov, Alexander V; Timofeev, Edward N

    2016-02-01

    Aptamers are nucleic acid-based scaffolds that can bind with high affinity to a variety of biological targets. Aptamers are identified from large DNA or RNA libraries through a process of directed molecular evolution (SELEX). Chemical modification of nucleic acids considerably increases the functional and structural diversity of aptamer libraries and substantially increases the affinity of the aptamers. Additionally, modified aptamers exhibit much greater resistance to biodegradation. The evolutionary selection of modified aptamers is conditioned by the possibility of the enzymatic synthesis and replication of non-natural nucleic acids. Wild-type or mutant polymerases and their non-natural nucleotide substrates that can support SELEX are highlighted in the present review. A focus is made on the efforts to find the most suitable type of nucleotide modifications and the engineering of new polymerases. Post-SELEX modification as a complementary method will be briefly considered as well.

  13. Aptamer contained triple-helix molecular switch for rapid fluorescent sensing of acetamiprid.

    Science.gov (United States)

    Liu, Xin; Li, Ying; Liang, Jing; Zhu, Wenyue; Xu, Jingyue; Su, Ruifang; Yuan, Lei; Sun, Chunyan

    2016-11-01

    In this study, an aptamer-based fluorescent sensing platform using triple-helix molecular switch (THMS) was developed for the pesticide screening represented by acetamiprid. The THMS was composed of two tailored DNA probes: a label-free central target specific aptamer sequence flanked by two arm segments acting as a recognition probe; a hairpin-shaped structure oligonucleotide serving as a signal transduction probe (STP), labeled with a fluorophore and a quencher at the 3' and 5'-end, respectively. In the absence of acetamiprid, complementary bindings of two arm segments of the aptamers with the loop sequence of STP enforce the formation of THMS with the "open" configuration of STP, and the fluorescence of THMS is on. In the presence of target acetamiprid, the aptamer-target binding results in the formation of a structured aptamer/target complex, which disassembles the THMS and releases the STP. The free STP is folded to a stem loop structure, and the fluorescence is quenched. The quenched fluorescence intensity was proportional to the concentration of acetamiprid in the range from 100 to 1200nM, with the limit of detection (LOD) as low as 9.12nM. In addition, this THMS-based method has been successfully used to test and quantify acetamiprid in Chinese cabbage with satisfactory recoveries, and the results were in full agreement with those from LC-MS. The aptamer-based THMS presents distinct advantages, including high stability, remarkable sensitivity, and preservation of the affinity and specificity of the original aptamer. Most importantly, this strategy is convenient and generalizable by virtue of altering the aptamer sequence without changing the triple-helix structure. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.

  14. Screening aptamers of cortisol by target-induced-structure-switching-dissociation SELEX%靶标诱导变构解离SELEX技术筛选皮质醇特异核酸适体

    Institute of Scientific and Technical Information of China (English)

    张岭; 王新兴; 战锐; 弓景波; 钱令嘉

    2011-01-01

    目的 通过指数富集配体系统进化(SELEX)技术获得皮质醇特异性核酸适体,建立高品质、超敏、易用和稳定的皮质醇检测试剂盒.方法 人工合成两端固定序列,中间为35 bp随机序列,生成80 bp长度ssDNA文库,捕获序列固定在磁珠上,文库与其退火组装,皮质醇的特异核酸适体通过我们建立的靶标诱导变构解离SELEX(TISSD-SELEX)技术获得.最后一轮的PCR产物经克隆测序,MegAlign 和RNAstructure软件分析其一级和二级结构.实时(real-time)apta-PCR方法分析G12的亲和活性.结果 经过12轮筛选后,获得10个皮质醇的核酸适体.序列分析显示,特异核酸适体以高G含量核酸序列为主,软件预测其中8个序列主要以G四联体二级结构为主,候选核酸适体 G12显示了可以作为生物探针用于皮质醇检测.结论 成功建立TISSD-SELEX技术,获得小分子靶标皮质醇核酸适体.小分子靶标无需将其偶联在固相介质或大分子上,对靶分子结构、性质无特殊要求,因而有望成为小分子靶标核酸适体筛选的通用技术方法.%Objective To obtain and identify aptamers which can specifically bind to cortisol by systematic evolution of ligands by exponential enrichment ( SELEX ) protocol,in order to establish a high-quality, ultra-sensitive, simple & robust cortisol detection kit. Methods A synthesized 80mer single stranded DNA random library containing 35 random sequences flanked by invariant primer was designed. Capture sequences were absorbed on the magnetic beads, the library and capture sequences were annealed and assembled, and the aptamers binding cortisol were obtained by the protocol which we had established as a screening method of target-induced-structure-switching-dissociation ( TISSD )-SELEX. PCR products of the last round selection were cloned and sequenced. MegAlign and RNAstructure software was employed to analyze the primary structure and secondary structures of the aptamers. Real

  15. Single-Round Patterned DNA Library Microarray Aptamer Lead Identification

    Directory of Open Access Journals (Sweden)

    Jennifer A. Martin

    2015-01-01

    Full Text Available A method for identifying an aptamer in a single round was developed using custom DNA microarrays containing computationally derived patterned libraries incorporating no information on the sequences of previously reported thrombin binding aptamers. The DNA library was specifically designed to increase the probability of binding by enhancing structural complexity in a sequence-space confined environment, much like generating lead compounds in a combinatorial drug screening library. The sequence demonstrating the highest fluorescence intensity upon target addition was confirmed to bind the target molecule thrombin with specificity by surface plasmon resonance, and a novel imino proton NMR/2D NOESY combination was used to screen the structure for G-quartet formation. We propose that the lack of G-quartet structure in microarray-derived aptamers may highlight differences in binding mechanisms between surface-immobilized and solution based strategies. This proof-of-principle study highlights the use of a computational driven methodology to create a DNA library rather than a SELEX based approach. This work is beneficial to the biosensor field where aptamers selected by solution based evolution have proven challenging to retain binding function when immobilized on a surface.

  16. Screening of Aptamers on Microfluidic Systems for Clinical Applications

    OpenAIRE

    Gwo-Bin Lee; Chao-Jyun Huang; Chen-Hsun Weng

    2012-01-01

    The use of microfluidic systems for screening of aptamers and their biomedical applications are reviewed in this paper. Aptamers with different nucleic acid sequences have been extensively studied and the results demonstrated a strong binding affinity to target molecules such that they can be used as promising candidate biomarkers for diagnosis and therapeutics. Recently, the aptamer screening protocol has been conducted with microfluidic-based devices. Furthermore, aptamer affinity screening...

  17. Strategies for the discovery of therapeutic Aptamers

    Science.gov (United States)

    Yang, Xianbin; Li, Na; Gorenstein, David G.

    2010-01-01

    Importance of the field Therapeutic aptamers are synthetic, structured oligonucleotides that bind to a very broad range of targets with high affinity and specificity. They are an emerging class of targeting ligand that show great promise for treating a number of diseases. A series of aptamers currently in various stages of clinical development highlights the potential of aptamers for therapeutic applications. Area covered in this review This review will cover in vitro selection of oligonucleotide ligands, called aptamers, from a combinatorial library using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process as well as the other known strategies for finding aptamers against various targets. What the reader will gain Readers will gain an understanding of the highly useful strategies for successful aptamer discovery. They may also be able combine two or more of the presented strategies for their aptamer discovery projects. Take home message Although many processes are available for discovering aptamers, it is not trivial to discover an aptamer candidate that is ready to move toward pharmaceutical drug development. It is also apparent that there have been relatively few therapeutic advances and clinical trials undertaken due to the small number of companies that participate in aptamer development. PMID:21359096

  18. Crystal structure of Hfq from Bacillus subtilis in complex with SELEX-derived RNA aptamer: insight into RNA-binding properties of bacterial Hfq

    Science.gov (United States)

    Someya, Tatsuhiko; Baba, Seiki; Fujimoto, Mai; Kawai, Gota; Kumasaka, Takashi; Nakamura, Kouji

    2012-01-01

    Bacterial Hfq is a protein that plays an important role in the regulation of genes in cooperation with sRNAs. Escherichia coli Hfq (EcHfq) has two or more sites that bind RNA(s) including U-rich and/or the poly(A) tail of mRNA. However, functional and structural information about Bacillus subtilis Hfq (BsHfq) including the RNA sequences that specifically bind to it remain unknown. Here, we describe RNA aptamers including fragment (AG)3A that are recognized by BsHfq and crystal structures of the BsHfq–(AG)3A complex at 2.2 Å resolution. Mutational and structural studies revealed that the RNA fragment binds to the distal site, one of the two binding sites on Hfq, and identified amino acid residues that are critical for sequence-specific interactions between BsHfq and (AG)3A. In particular, R32 appears to interact with G bases in (AG)3A. Poly(A) also binds to the distal site of EcHfq, but the overall RNA structure and protein–RNA interaction patterns engaged in the R32 residues of BsHfq–(AG)3A differ from those of EcHfq–poly(A). These findings provide novel insight into how the Hfq homologue recognizes RNA. PMID:22053080

  19. Functional detection of proteins by caged aptamers.

    Science.gov (United States)

    Pinto, Alessandro; Lennarz, Sabine; Rodrigues-Correia, Alexandre; Heckel, Alexander; O'Sullivan, Ciara K; Mayer, Günter

    2012-02-17

    While many diagnostic assay platforms enable the measurement of analytes with high sensitivity, most of them result in a disruption of the analyte's native structure and, thus, in loss of function. Consequently, the analyte can be used neither for further analytical assessment nor functional analysis. Herein we report the use of caged aptamers as templates during apta-PCR analysis of targets. Aptamers are short nucleic acids that fold into a well-defined three-dimensional structure in which they interact with target molecules with high affinity and specificity. Nucleic acid aptamers can also serve as templates for qPCR approaches and, thus, have been used as high affinity ligands to bind to target molecules and subsequently for quantification by qPCR, an assay format coined apta-PCR. Caged aptamers in turn refer to variants that bear one or more photolabile groups at strategic positions. The activity of caged aptamers can thus be turned on or off by light irradiation. The latter allows the mild elution of target-bound aptamers while the target's native structure and function remain intact. We demonstrate that this approach allows the quantitative and subsequently the functional assessment of analytes. Since caged aptamers can be generated emanating from virtually every available aptamer, the described approach can be generalized and adopted to any target-aptamer pair and, thus, have a broad applicability in proteomics and clinical diagnostics.

  20. Exploring the binding of d(GGGT)4 to the HIV-1 integrase: An approach to investigate G-quadruplex aptamer/target protein interactions.

    Science.gov (United States)

    Esposito, Veronica; Pirone, Luciano; Mayol, Luciano; Pedone, Emilia; Virgilio, Antonella; Galeone, Aldo

    2016-08-01

    The aptamer d(GGGT)4 (T30923 or T30695) forms a 5'-5' dimer of two stacked parallel G-quadruplexes, each characterized by three G-tetrads and three single-thymidine reversed-chain loops. This aptamer has been reported to exhibit anti-HIV activity by targeting the HIV integrase, a viral enzyme responsible for the integration of viral DNA into the host-cell genome. However, information concerning the aptamer/target interaction is still rather limited. In this communication we report microscale thermophoresis investigations on the interaction between the HIV-1 integrase and d(GGGT)4 aptamer analogues containing abasic sites singly replacing thymidines in the original sequence. This approach has allowed the identification of which part of the aptamer G-quadruplex structure is mainly involved in the interaction with the protein.

  1. Quinine binding by the cocaine-binding aptamer. Thermodynamic and hydrodynamic analysis of high-affinity binding of an off-target ligand.

    Science.gov (United States)

    Reinstein, Oren; Yoo, Mina; Han, Chris; Palmo, Tsering; Beckham, Simone A; Wilce, Matthew C J; Johnson, Philip E

    2013-12-03

    The cocaine-binding aptamer is unusual in that it tightly binds molecules other than the ligand it was selected for. Here, we study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titration calorimetry was used to quantify the quinine-binding affinity and thermodynamics of a set of sequence variants of the cocaine-binding aptamer. We find that the affinity of the cocaine-binding aptamer for quinine is 30-40 times stronger than it is for cocaine. Competitive-binding studies demonstrate that both quinine and cocaine bind at the same site on the aptamer. The ligand-induced structural-switching binding mechanism of an aptamer variant that contains three base pairs in stem 1 is retained with quinine as a ligand. The short stem 1 aptamer is unfolded or loosely folded in the free form and becomes folded when bound to quinine. This folding is confirmed by NMR spectroscopy and by the short stem 1 construct having a more negative change in heat capacity of quinine binding than is seen when stem 1 has six base pairs. Small-angle X-ray scattering (SAXS) studies of the free aptamer and both the quinine- and the cocaine-bound forms show that, for the long stem 1 aptamers, the three forms display similar hydrodynamic properties, and the ab initio shape reconstruction structures are very similar. For the short stem 1 aptamer there is a greater variation among the SAXS-derived ab initio shape reconstruction structures, consistent with the changes expected with its structural-switching binding mechanism.

  2. Crystal structure of a mirror-image L-RNA aptamer (Spiegelmer) in complex with the natural L-protein target CCL2

    Science.gov (United States)

    Oberthür, Dominik; Achenbach, John; Gabdulkhakov, Azat; Buchner, Klaus; Maasch, Christian; Falke, Sven; Rehders, Dirk; Klussmann, Sven; Betzel, Christian

    2015-04-01

    We report the crystal structure of a 40mer mirror-image RNA oligonucleotide completely built from nucleotides of the non-natural L-chirality in complex with the pro-inflammatory chemokine L-CLL2 (monocyte chemoattractant protein-1), a natural protein composed of regular L-amino acids. The L-oligonucleotide is an L-aptamer (a Spiegelmer) identified to bind L-CCL2 with high affinity, thereby neutralizing the chemokine's activity. CCL2 plays a key role in attracting and positioning monocytes; its overexpression in several inflammatory diseases makes CCL2 an interesting pharmacological target. The PEGylated form of the L-aptamer, NOX-E36 (emapticap pegol), already showed promising efficacy in clinical Phase II studies conducted in diabetic nephropathy patients. The structure of the L-oligonucleotide.L-protein complex was solved and refined to 2.05 Å. It unveils the L-aptamer's intramolecular contacts and permits a detailed analysis of its structure-function relationship. Furthermore, the analysis of the intermolecular drug-target interactions reveals insight into the selectivity of the L-aptamer for certain related chemokines.

  3. DNA aptamers for selective identification and separation of flame retardant chemicals.

    Science.gov (United States)

    Kim, Un-Jung; Kim, Byoung Chan

    2016-09-14

    Polybrominated diphenyl ethers (PBDEs) are group of chemicals which are representative persistent organic pollutants (POPs) and used as brominated flame retardants for many consumer products. PBDEs were phased out since 2009 but are still frequently observed in various environmental matrices and human body. Here, we report ssDNA aptamers which bind to BDE47, one of the PBDE congeners commonly found in various environmental matrices, and show affinity to other major tri-to hepta- BDE congeners. The PBDE specific aptamers were isolated from random library of ssDNA using Mag-SELEX. Two out of 15 sequences, based on their alignment and hairpin loop structures, were chosen to determine dissociation constant with BDE47 and showed from picomolar to nanomolar affinities (200 pM and 1.53 nM). The aptamers displayed high selectivity to the original target, BDE47, and implying general specificity to PBDE backbone with varying affinities to other congeners. Further, we showed that the use of two aptamers together could enhance the separation efficiency of BDE47 and other BDE congeners when dissolved in a solvent compared to use of single aptamer. These aptamers are expected to provide a tool for preliminary screening or quick separation of PBDEs in environmental samples prior to trace quantitative analysis.

  4. FRET-Aptamer Assays for Bone Marker Assessment, C-Telopeptide, Creatinine, and Vitamin D

    Science.gov (United States)

    Bruno, John G.

    2013-01-01

    Astronauts lose 1.0 to 1.5% of their bone mass per month on long-duration spaceflights. NASA wishes to monitor the bone loss onboard spacecraft to develop nutritional and exercise countermeasures, and make adjustments during long space missions. On Earth, the same technology could be used to monitor osteoporosis and its therapy. Aptamers bind to targets against which they are developed, much like antibodies. However, aptamers do not require animal hosts or cell culture and are therefore easier, faster, and less expensive to produce. In addition, aptamers sometimes exhibit greater affinity and specificity vs. comparable antibodies. In this work, fluorescent dyes and quenchers were added to the aptamers to enable pushbutton, one-step, bind-and-detect fluorescence resonance energy transfer (FRET) assays or tests that can be freeze-dried, rehydrated with body fluids, and used to quantitate bone loss of vitamin D levels with a handheld fluorometer in the spacecraft environment. This work generated specific, rapid, one-step FRET assays for the bone loss marker C-telopeptide (CTx) when extracted from urine, creatinine from urine, and vitamin D congeners in diluted serum. The assays were quantified in nanograms/mL using a handheld fluorometer connected to a laptop computer to convert the raw fluorescence values into concentrations of each analyte according to linear standard curves. DNA aptamers were selected and amplified for several rounds against a 26- amino acid form of CTx, creatinine, and vitamin D. The commonalities between loop structures were studied, and several common loop structures were converted into aptamer beacons with a fluorophore and quencher on each end. In theory, when the aptamer beacon binds its cognate target (CTx bone peptide, creatinine, or vitamin D), it is forced open and no longer quenched, so it gives off fluorescent light (when excited) in proportion to the amount of target present in a sample. This proportional increase in fluorescence is

  5. Aptamers: Biomedical Interest and Applications

    Directory of Open Access Journals (Sweden)

    Cristina Romero-López

    2017-03-01

    Full Text Available Aptamers are short DNA or RNA oligonucleotides specialized in the specific and efficient binding to a target molecule. They are obtained by in vitro selection or evolution processes. It was in 1990 that two independent research groups described the bases of a new in vitro technology for the identification of RNA molecules able to specifically bind to a target [1,2]. Tuerk and Gold established the principals of the in vitro selection process that was named SELEX (Systematic Evolution of Ligands by Exponential enrichment, which is based on iterative cycles of binding, partitioning, and amplification of oligonucleotides from a pool of variant sequences [2]. Ellington and Szostak coined the term aptamer to define the selected molecules by the application of this method [1]. To date, numerous reports have described the isolation of aptamers directed against a great variety of targets covering a wide diversity of molecules varying in nature, size, and complexity ranging from ions to whole cells, including small molecules (e.g., aminoacids, nucleotides, antibiotics, peptides, proteins, nucleic acids, and viruses, among others (for example, see [3–6]. Modifications and optimization of the SELEX procedure aimed to get newly modified aptamers has also attracted much interest (examples can be found in [7,8]. These advances along with the parallel progresses in the nucleic acids chemistry and cellular delivery fields have allowed for the rise of a new hope in developing aptamers as efficient molecular tools for diagnostics and therapeutics (for recent comprehensive reviews, see [9–11].

  6. Enhancing sensitivity and selectivity of long-period grating sensors using structure-switching aptamers bound to gold-doped macroporous silica coatings.

    Science.gov (United States)

    Carrasquilla, Carmen; Xiao, Yao; Xu, Chang Qing; Li, Yingfu; Brennan, John D

    2011-10-15

    High surface area, sol-gel derived macroporous silica films doped with gold nanoparticles (AuNP) are used as a platform for high-density affinity-based immobilization of functional structure-switching DNA aptamer molecules onto Michelson interferometer long-period grating (LPG) fiber sensors, allowing for label-free detection of small molecular weight analytes such as adenosine triphosphate (ATP). The high surface area afforded by the sol-gel derived material allowed high loading of DNA aptamers, while the inclusion of gold nanoparticles within the silica film provided a high refractive index (RI) overlay, which is required to enhance the sensitivity of the LPG sensor according to our numerical simulations. By using a structure-switching aptamer construct that could release an oligonucleotide upon binding of ATP, the effective change in RI was both enhanced and inverted (i.e., binding of ATP caused a net reduction in molecular weight and refractive index), resulting in a system that prevented signals originating from nonspecific binding. This is the first report on the coupling of aptamers to LPG fiber sensors and the first use of high RI AuNP/silica films as supports to immobilize biomolecules onto the LPG sensor surface. The dual functionality of such films to both improve binding density and LPG sensor cladding refractive index results in a substantial enhancement in the sensitivity of such sensors for small molecule detection.

  7. Discrimination of recombinant from natural human growth hormone using DNA aptamers.

    Science.gov (United States)

    Bruno, John G; Carrillo, Maria P; Phillips, Taylor; Edge, Allison

    2011-04-01

    Detection of athletes who use synthetic human growth hormone (hGH; or somatotropin) to enhance physical strength and obtain an advantage in competitive sports is a formidable problem, as rhGH is virtually identical to the natural pituitary hormone. However, some post-translational and other modifications have been documented by chromatographic separation and mass spectrometry (MS) in a small percentage of rhGH. In the present work, development of DNA aptamers against research-grade rhGH and natural hGH with adsorption of the rhGH aptamers against natural hGH was shown to produce a small family of aptamer sequences that bound consistently with greater affinity to rhGH over a low nanogram-to-microgram range in ELISA-like microplate assays. This collection of rhGH discriminatory aptamer sequences shared some short sequence segments and secondary structural features. The top rhGH discriminatory aptamers also appeared to cross-react with human myoglobin and BSA but not with bone collagen peptides and an unrelated viral envelope peptide. The cross-reactivity results suggested several strings of up to five consecutive amino acids that might serve as common epitopes for aptamer binding. SDS-PAGE revealed that the rhGH existed largely as a 45-kDa dimer, and the natural hGH was almost exclusively monomeric. The existence of the rhGH dimer suggests that a discontinuous "bridge" epitope may exist on the rhGH, which spans the subunits, thereby accounting somewhat for the difference in detection. Overall, these results suggest that aptamers might be useful for routine, presumptive laboratory screening to identify athletes who are potentially cheating by administration of rhGH.

  8. Highly efficient inhibition of human immunodeficiency virus type 1 reverse transcriptase by aptamers functionalized gold nanoparticles

    Science.gov (United States)

    Shiang, Yen-Chun; Ou, Chung-Mao; Chen, Shih-Ju; Ou, Ting-Yu; Lin, Han-Jia; Huang, Chih-Ching; Chang, Huan-Tsung

    2013-03-01

    We have developed aptamer (Apt)-conjugated gold nanoparticles (Apt-Au NPs, 13 nm in diameter) as highly effective inhibitors for human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Two Apts, RT1t49 (Aptpol) and ODN 93 (AptRH), which recognize the polymerase and RNase H regions of HIV-1 RT, are used to conjugate Au NPs to prepare Aptpol-Au NPs and AptRH-Au NPs, respectively. In addition to DNA sequence, the surface density of the aptamers on Au NPs (nApt-Au NPs; n is the number of aptamer molecules on each Au NP) and the linker length number (Tm; m is the base number of the deoxythymidine linker) between the aptamer and Au NPs play important roles in determining their inhibition activity. A HIV-lentiviral vector-based antiviral assay has been applied to determine the inhibitory effect of aptamers or Apt-Au NPs on the early stages of their replication cycle. The nuclease-stable G-quadruplex structure of 40AptRH-T45-Au NPs shows inhibitory efficiency in the retroviral replication cycle with a decreasing infectivity (40.2%).We have developed aptamer (Apt)-conjugated gold nanoparticles (Apt-Au NPs, 13 nm in diameter) as highly effective inhibitors for human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Two Apts, RT1t49 (Aptpol) and ODN 93 (AptRH), which recognize the polymerase and RNase H regions of HIV-1 RT, are used to conjugate Au NPs to prepare Aptpol-Au NPs and AptRH-Au NPs, respectively. In addition to DNA sequence, the surface density of the aptamers on Au NPs (nApt-Au NPs; n is the number of aptamer molecules on each Au NP) and the linker length number (Tm; m is the base number of the deoxythymidine linker) between the aptamer and Au NPs play important roles in determining their inhibition activity. A HIV-lentiviral vector-based antiviral assay has been applied to determine the inhibitory effect of aptamers or Apt-Au NPs on the early stages of their replication cycle. The nuclease-stable G-quadruplex structure of 40AptRH-T45

  9. Assessing the amount of quadruplex structures present within G₂-tract synthetic random-sequence DNA libraries.

    Science.gov (United States)

    McManus, Simon A; Li, Yingfu

    2013-01-01

    The process of in vitro selection has led to the discovery of many aptamers with potential to be developed into inhibitors and biosensors, but problems in isolating aptamers against certain targets with desired affinity and specificity still remain. One possible improvement is to use libraries enhanced for motifs repeatedly isolated in aptamer molecules. One such frequently observed motif is the two-tiered guanine quadruplex. In this study we investigated whether DNA libraries could be designed to contain a large fraction of molecules capable of folding into two-tiered guanine quadruplexes. Using comprehensive circular dichroism analysis, we found that DNA libraries could be designed to contain a large proportion of sequences that adopt guanine quadruplex structures. Analysis of individual sequences from a small library revealed a mixture of quadruplexes of different topologies providing the diversity desired for an in vitro selection. We also found that primer-binding sites are detrimental to quadruplex formation and devised a method for post-selection amplification of primer-less quadruplex libraries. With the development of guanine quadruplex enriched DNA libraries, it should be possible to improve the chances of isolating aptamers that utilize a quadruplex scaffold and enhance the success of in vitro selection experiments.

  10. Mapping the affinity landscape of Thrombin-binding aptamers on 2'F-ANA/DNA chimeric G-Quadruplex microarrays.

    Science.gov (United States)

    Lietard, Jory; Abou Assi, Hala; Gómez-Pinto, Irene; González, Carlos; Somoza, Mark M; Damha, Masad J

    2017-01-18

    In situ fabricated nucleic acids microarrays are versatile and very high-throughput platforms for aptamer optimization and discovery, but the chemical space that can be probed against a given target has largely been confined to DNA, while RNA and non-natural nucleic acid microarrays are still an essentially uncharted territory. 2'-Fluoroarabinonucleic acid (2'F-ANA) is a prime candidate for such use in microarrays. Indeed, 2'F-ANA chemistry is readily amenable to photolithographic microarray synthesis and its potential in high affinity aptamers has been recently discovered. We thus synthesized the first microarrays containing 2'F-ANA and 2'F-ANA/DNA chimeric sequences to fully map the binding affinity landscape of the TBA1 thrombin-binding G-quadruplex aptamer containing all 32 768 possible DNA-to-2'F-ANA mutations. The resulting microarray was screened against thrombin to identify a series of promising 2'F-ANA-modified aptamer candidates with Kds significantly lower than that of the unmodified control and which were found to adopt highly stable, antiparallel-folded G-quadruplex structures. The solution structure of the TBA1 aptamer modified with 2'F-ANA at position T3 shows that fluorine substitution preorganizes the dinucleotide loop into the proper conformation for interaction with thrombin. Overall, our work strengthens the potential of 2'F-ANA in aptamer research and further expands non-genomic applications of nucleic acids microarrays.

  11. A novel electrochemical aptasensor based on Y-shape structure of dual-aptamer-complementary strand conjugate for ultrasensitive detection of myoglobin.

    Science.gov (United States)

    Taghdisi, Seyed Mohammad; Danesh, Noor Mohammad; Ramezani, Mohammad; Emrani, Ahmad Sarreshtehdar; Abnous, Khalil

    2016-06-15

    Monitoring of myoglobin (Mb) in human blood serum is highly in demand for early diagnosis of acute myocardial infarction (AMI). Here, a novel electrochemical aptasensor was developed for ultrasensitive and selective detection of Mb, based on Y-shape structure of dual-aptamer (DApt)-complementary strand of aptamer (CS) conjugate, gold electrode and exonuclease I (Exo I). The designed aptasensor obtains features of gold, such as high electrochemical conductivity and large surface area, property of Y-shape structure of DApt-CS conjugate to function as a gate and obstacle for the access of redox probe to the surface of electrode, as well as high specificity and sensitivity of aptamer toward its target and Exo I as an enzyme which specifically degrades the 3'-end of single-stranded DNA (ssDNA). In the absence of Mb, the Y-shape structure remains intact. So, a weak electrochemical signal is observed. Upon addition of target, the DApt leave the CS and bind to Mb, leading to disassembly of Y-shape structure and following the addition of Exo I, a strong electrochemical signal could be recorded. The fabricated aptasensor showed high selectivity toward Mb with a limit of detection (LOD) as low as 27 pM. Besides, the developed aptasensor was effectively applied to detect Mb in human serum.

  12. Development of an Antigen-DNAzyme Based Probe for a Direct Antibody-Antigen Assay Using the Intrinsic DNAzyme Activity of a Daunomycin Aptamer

    Directory of Open Access Journals (Sweden)

    Noorsharmimi Omar

    2013-12-01

    Full Text Available G-Quadruplex (G-4 structures are formed when G-rich DNA sequences fold into intra- or intermolecular four-stranded structures in the presence of metal ions. G-4-hemin complexes are often effective peroxidase-mimicking DNAzymes that are applied in many detection systems. This work reports the application of a G-rich daunomycin-specific aptamer for the development of an antibody-antigen detection assay. We investigated the ability of the daunomycin aptamer to efficiently catalyze the hemin-dependent peroxidase activity independent of daunomycin. A reporter probe consisting of biotinylated antigen and daunomycin aptamer coupled to streptavidin gold nanoparticles was successfully used to generate a colorimetric readout. In conclusion, the daunomycin aptamer can function as a robust alternative DNAzyme for the development of colorimetric assays.

  13. A novel electrochemical aptasensor based on arch-shape structure of aptamer-complimentary strand conjugate and exonuclease I for sensitive detection of streptomycin.

    Science.gov (United States)

    Mohammad Danesh, Noor; Ramezani, Mohammad; Sarreshtehdar Emrani, Ahmad; Abnous, Khalil; Taghdisi, Seyed Mohammad

    2016-01-15

    Detection and quantitation of antibiotic residues in blood serum and animal foodstuffs are of great significance. In this study, an electrochemical aptasensor was developed for sensitive and selective detection of streptomycin, based on exonuclease I (Exo I), complimentary strand of aptamer (CS), Arch-shape structure of aptamer (Apt)-CS conjugate and gold electrode. The designed aptasensor inherits characteristics of gold including large surface area and high electrochemical conductivity, as well as high sensitivity and selectivity of aptamer toward its target, property of Arch-shape structure of Apt-CS conjugate to act as a gate and barrier for the access of redox probe to the surface of electrode and the function of Exo I as an enzyme which selectively digests the 3'-end of single stranded DNA (ssDNA). In the absence of streptomycin the gate remains closed. Thus, the electrochemical signal is weak. Upon addition of streptomycin, the Apt leaves the CS and binds to streptomycin and the Arch-shape structure is disassembled. Then, Exo I addition leads to a strong electrochemical signal. The designed electrochemical aptasensor exhibited high selectivity toward streptomycin with a limit of detection (LOD) as low as 11.4nM. Moreover, the developed electrochemical aptasensor was successfully used to detect streptomycin in milk and serum with LODs of 14.1 and 15.3nM, respectively.

  14. A novel fluorescent aptasensor based on hairpin structure of complementary strand of aptamer and nanoparticles as a signal amplification approach for ultrasensitive detection of cocaine.

    Science.gov (United States)

    Emrani, Ahmad Sarreshtehdar; Danesh, Noor Mohammad; Ramezani, Mohammad; Taghdisi, Seyed Mohammad; Abnous, Khalil

    2016-05-15

    Cocaine is one of the most commonly misused stimulant which could influence the central nervous system. In this study, a fluorescent aptamer-based sensor (aptasensor) was designed for sensitive and selective detection of cocaine, based on hairpin structure of complementary strand of aptamer (CS), target-induced release of aptamer (Apt) from CS and two kinds of nanoparticles, including silica nanoparticles (SNPs) coated with streptavidin and gold nanoparticles (AuNPs). The designed aptasensor acquires characteristics of AuNPs such as unique optical properties and large surface area, SNPs as amplifiers of fluorescence intensity, higher affinity of Apt toward its target relative to its CS, and finally the hairpin structure of CS that brings the fluorophore (FAM) to close proximity to the surface of SNPs. In the absence of cocaine, FAM is in close proximity to the surface of AuNPs, resulting in a weak fluorescence emission. In the presence of target, FAM comes to close proximity to the surface of SNPs because of the formation of hairpin structure of CS, leading to a very strong fluorescence emission. The fabricated fluorescent aptasensor exhibited a good selectivity toward cocaine with a limit of detection (LOD) as low as 209 pM. Moreover, the designed aptasensor was successfully utilized to detect cocaine in serum with a LOD as low as 293 pM.

  15. The structural basis for recognition of the PreQ0 metabolite by an unusually small riboswitch aptamer domain.

    Science.gov (United States)

    Spitale, Robert C; Torelli, Andrew T; Krucinska, Jolanta; Bandarian, Vahe; Wedekind, Joseph E

    2009-04-24

    Riboswitches are RNA elements that control gene expression through metabolite binding. The preQ(1) riboswitch exhibits the smallest known ligand-binding domain and is of interest for its economical organization and high affinity interactions with guanine-derived metabolites required to confer tRNA wobbling. Here we present the crystal structure of a preQ(1) aptamer domain in complex with its precursor metabolite preQ(0). The structure is highly compact with a core that features a stem capped by a well organized decaloop. The metabolite is recognized within a deep pocket via Watson-Crick pairing with C15. Additional hydrogen bonds are made to invariant bases U6 and A29. The ligand-bound state confers continuous helical stacking throughout the core fold, thus providing a platform to promote Watson-Crick base pairing between C9 of the decaloop and the first base of the ribosome-binding site, G33. The structure offers insight into the mode of ribosome-binding site sequestration by a minimal RNA fold stabilized by metabolite binding and has implications for understanding the molecular basis by which bacterial genes are regulated.

  16. Anti-HCV RNA Aptamers Targeting the Genomic cis-Acting Replication Element

    Directory of Open Access Journals (Sweden)

    Alfredo Berzal-Herranz

    2011-12-01

    Full Text Available Hepatitis C virus (HCV replication is dependent on the existence of several highly conserved functional genomic RNA domains. The cis-acting replication element (CRE, located within the 3' end of the NS5B coding region of the HCV genome, has been shown essential for efficient viral replication. Its sequence and structural features determine its involvement in functional interactions with viral RNA-dependent RNA polymerase and distant RNA domains of the viral genome. This work reports the use of an in vitro selection strategy to select aptamer RNA molecules against the complete HCV-CRE. After six selection cycles, five potential target sites were identified within this domain. Inhibition assays using a sample of representative aptamers showed that the selected RNAs significantly inhibit the replication (>80% of a subgenomic HCV replicon in Huh-7 cell cultures. These results highlight the potential of aptamer RNA molecules as therapeutic antiviral agents.

  17. Nucleic acid aptamers: research tools in disease diagnostics and therapeutics.

    Science.gov (United States)

    Santosh, Baby; Yadava, Pramod K

    2014-01-01

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

  18. Optimized light-directed synthesis of aptamer microarrays.

    Science.gov (United States)

    Franssen-van Hal, Nicole L W; van der Putte, Pepijn; Hellmuth, Klaus; Matysiak, Stefan; Kretschy, Nicole; Somoza, Mark M

    2013-06-18

    Aptamer microarrays are a promising high-throughput method for ultrasensitive detection of multiple analytes, but although much is known about the optimal synthesis of oligonucleotide microarrays used in hybridization-based genomics applications, the bioaffinity interactions between aptamers and their targets is qualitatively different and requires significant changes to synthesis parameters. Focusing on streptavidin-binding DNA aptamers, we employed light-directed in situ synthesis of microarrays to analyze the effects of sequence fidelity, linker length, surface probe density, and substrate functionalization on detection sensitivity. Direct comparison with oligonucleotide hybridization experiments indicates that aptamer microarrays are significantly more sensitive to sequence fidelity and substrate functionalization and have different optimal linker length and surface probe density requirements. Whereas microarray hybridization probes generate maximum signal with multiple deletions, aptamer sequences with the same deletion rate result in a 3-fold binding signal reduction compared with the same sequences synthesized for maximized sequence fidelity. The highest hybridization signal was obtained with dT 5mer linkers, and the highest aptamer signal was obtained with dT 11mers, with shorter aptamer linkers significantly reducing the binding signal. The probe hybridization signal was found to be more sensitive to molecular crowding, whereas the aptamer probe signal does not appear to be constrained within the density of functional surface groups commonly used to synthesize microarrays.

  19. Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy.

    Science.gov (United States)

    Ruff, Karen M; Strobel, Scott A

    2014-11-01

    The glycine riboswitch predominantly exists as a tandem structure, with two adjacent, homologous ligand-binding domains (aptamers), followed by a single expression platform. The recent identification of a leader helix, the inclusion of which eliminates cooperativity between the aptamers, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch. An equilibrium dialysis-based assay was combined with binding-site mutations to monitor glycine binding in each ligand-binding site independently to understand the role of each aptamer in glycine binding and riboswitch tertiary interactions. A series of mutations disrupting the dimer interface was used to probe how dimerization impacts ligand binding by the tandem glycine riboswitch. While the wild-type tandem riboswitch binds two glycine equivalents, one for each aptamer, both individual aptamers are capable of binding glycine when the other aptamer is unoccupied. Intriguingly, glycine binding by aptamer-1 is more sensitive to dimerization than glycine binding by aptamer-2 in the context of the tandem riboswitch. However, monomeric aptamer-2 shows dramatically weakened glycine-binding affinity. In addition, dimerization of the two aptamers in trans is dependent on glycine binding in at least one aptamer. We propose a revised model for tandem riboswitch function that is consistent with these results, wherein ligand binding in aptamer-1 is linked to aptamer dimerization and stabilizes the P1 stem of aptamer-2, which controls the expression platform.

  20. Serum inverts and improves the fluorescence response of an aptamer beacon to various vitamin D analytes.

    Science.gov (United States)

    Bruno, John G; Carrillo, Maria P; Phillips, Taylor; Edge, Allison

    2012-01-01

    A dominant aptamer loop structure from a library of nearly 100 candidate aptamer sequences developed against immobilized 25-hydroxyvitamin D(3) (calcidiol) was converted into a 5'-TYE 665 and 3'-Iowa black-labelled aptamer beacon. The aptamer beacon exhibited a mild 'lights on' reaction in buffer as a function of increasing concentrations of several vitamin D analogues and metabolites, with a limit of detection of approximately 200 ng/mL, and was not specific for any particular congener. In 10% or 50% human serum, the same aptamer beacon inverted its fluorescence behaviour to become a more intense 'lights off' reaction with an improved limit of detection in the range 4-16 ng/mL. We hypothesized that this drastic change in fluorescence behaviour was due to the presence of creatinine and urea in serum, which might destabilize the quenched beacon, causing an increase in fluorescence followed by decreasing fluorescence as a function of vitamin D concentrations that may bind and quench increasingly greater fractions of the denatured beacons. However, the results of several control experiments in the presence of physiological or greater concentrations of creatinine and urea, alone or combined in buffer, failed to produce the beacon fluorescence inversion. Other possible mechanistic hypotheses are also discussed.

  1. Remyelination induced by a DNA aptamer in a mouse model of multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Branislav Nastasijevic

    Full Text Available Multiple sclerosis (MS is a debilitating inflammatory disease of the central nervous system (CNS characterized by local destruction of the insulating myelin surrounding neuronal axons. With more than 200 million MS patients worldwide, the absence of treatments that prevent progression or induce repair poses a major challenge. Anti-inflammatory therapies have met with limited success only in preventing relapses. Previous screening of human serum samples revealed natural IgM antibodies that bind oligodendrocytes and promote both cell signaling and remyelination of CNS lesions in an MS model involving chronic infection of susceptible mice by Theiler's encephalomyelitis virus and in the lysolecithin model of focal demyelination. This intriguing result raises the possibility that molecules with binding specificity for oligodendrocytes or myelin components may promote therapeutic remyelination in MS. Because of the size and complexity of IgM antibodies, it is of interest to identify smaller myelin-specific molecules with the ability to promote remyelination in vivo. Here we show that a 40-nucleotide single-stranded DNA aptamer selected for affinity to murine myelin shows this property. This aptamer binds multiple myelin components in vitro. Peritoneal injection of this aptamer results in distribution to CNS tissues and promotes remyelination of CNS lesions in mice infected by Theiler's virus. Interestingly, the selected DNA aptamer contains guanosine-rich sequences predicted to induce folding involving guanosine quartet structures. Relative to monoclonal antibodies, DNA aptamers are small, stable, and non-immunogenic, suggesting new possibilities for MS treatment.

  2. Portable Detection of Melamine in Milk Using a Personal Glucose Meter Based on an in Vitro Selected Structure-Switching Aptamer.

    Science.gov (United States)

    Gu, Chunmei; Lan, Tian; Shi, Hanchang; Lu, Yi

    2015-08-04

    Melamine detection in milk and other foods has attracted much attention since the discovery that melamine-adulterated food causes severe kidney damage. Although many methods have been developed to detect melamine, few methods can provide quantitative results using an affordable and portable device that is suitable for home use or field application. To achieve this goal, we herein report the first in vitro selection of a melamine responsive aptamer using a structure-switching method. A personal glucose meter (PGM) based melamine sensor was designed and subsequently tested using the newly isolated aptamer. Conversion of melamine concentration to glucose amount was achieved by including an invertase-conjugated DNA that is complementary to part of the aptamer. Melamine binding triggers the release of the invertase-DNA conjugate, which hydrolyzes sucrose into glucose. The glucose produced is then measured directly using an off-the-shelf PGM. The described sensor shows high selectivity for melamine against several closely related melamine analogues, such as cyanuric acid, ammeline, and ammelide, and has low detection limits of 0.33 μM (or 41.1 ppb) in buffer and 0.53 μM (or 67.5 ppb) in 80% whole milk without any pretreatment. The detection limits meet the threshold of 2.5 ppm for non-infant-formula products and 1 ppm for melamine in infant milk products as defined by the U.S. Food and Drug Administration (FDA). In addition to the PGM sensor demonstrated here, the same aptamer can be converted into other types of sensors with different signal outputs, allowing portable detection of melamine under a variety of conditions.

  3. Aptamers for biosensors

    OpenAIRE

    Bini, A

    2009-01-01

    Aptamers are single-stranded DNA or RNA molecules isolated in vitro by a selection and amplification method. Aptamers bind with high specificity and affinity to a wide range of target molecules, with dissociation constant comparable to antibodies. In this work aptamers were employed as a new kind of bio-recognition element in affinity biosensors for the detection of clinically relevant proteins in heterogeneous assay, using Piezoelectric Quartz Crystal Microbalance and Surface ...

  4. Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics.

    Science.gov (United States)

    Liberman, Joseph A; Suddala, Krishna C; Aytenfisu, Asaminew; Chan, Dalen; Belashov, Ivan A; Salim, Mohammad; Mathews, David H; Spitale, Robert C; Walter, Nils G; Wedekind, Joseph E

    2015-07-07

    PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3' RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼ 0.6 s(-1)) and undocking (kundock ∼ 1.1 s(-1)). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs.

  5. Sequence-structure relations of biopolymers

    CERN Document Server

    Barrett, Christopher; Reidys, Christian M

    2015-01-01

    Motivation: DNA data is transcribed into single-stranded RNA, which folds into specific molecular structures. In this paper we pose the question to what extent sequence- and structure-information correlate. We view this correlation as structural semantics of sequence data that allows for a different interpretation than conventional sequence alignment. Structural semantics could enable us to identify more general embedded "patterns" in DNA and RNA sequences. Results: We compute the partition function of sequences with respect to a fixed structure and connect this computation to the mutual information of a sequence-structure pair for RNA secondary structures. We present a Boltzmann sampler and obtain the a priori probability of specific sequence patterns. We present a detailed analysis for the three PDB-structures, 2JXV (hairpin), 2N3R (3-branch multi-loop) and 1EHZ (tRNA). We localize specific sequence patterns, contrast the energy spectrum of the Boltzmann sampled sequences versus those sequences that refold ...

  6. Identification of ssDNA aptamers specific to clinical isolates of Streptococcus mutans strains with different cariogenicity.

    Science.gov (United States)

    Cui, Wei; Liu, Jiaojiao; Su, Donghua; Hu, Danyang; Hou, Shuai; Hu, Tongnan; Yang, Jiyong; Luo, Yanping; Xi, Qing; Chu, Bingfeng; Wang, Chenglong

    2016-06-01

    Streptococcus mutans, a Gram-positive facultative anaerobic bacterium, is considered to be a major etiological factor for dental caries. In this study, plaques from dental enamel surfaces of caries-active and caries-free individuals were obtained and cultivated for S. mutans isolation. Morphology examination, biochemical characterization, and polymerase chain reaction were performed to identify S. mutans The cariogenicity of S. mutans strains isolated from clinical specimens was evaluated by testing the acidogenicity, aciduricity, extracellular polysaccharide production, and adhesion ability of the bacteria. Finally, subtractive SELEX (systematic evolution of ligands by exponential enrichment) technology targeting whole intact cells was used to screen for ssDNA aptamers specific to the strains with high cariogenicity. After nine rounds of subtractive SELEX, sufficient pool enrichment was achieved as shown by radioactive isotope analysis. The enriched pool was cloned and sequenced randomly, followed by MEME online and RNA structure software analysis of the sequences. Results from the flow cytometry indicated that aptamers H1, H16, H4, L1, L10, and H19 could discriminate highly cariogenic S. mutans strains from poorly cariogenic strains. Among these, Aptamer H19 had the strongest binding capacity with cariogenic S. mutans strains with a dissociation constant of 69.45 ± 38.53 nM. In conclusion, ssDNA aptamers specific to highly cariogenic clinical S. mutans strains were successfully obtained. These ssDNA aptamers might be used for the early diagnosis and treatment of dental caries.

  7. Aptamers: A Feasible Technology in Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    M. M. Soldevilla

    2016-01-01

    Full Text Available Aptamers are single-chained RNA or DNA oligonucleotides (ODNs with three-dimensional folding structures which allow them to bind to their targets with high specificity. Aptamers normally show affinities comparable to or higher than that of antibodies. They are chemically synthesized and therefore less expensive to manufacture and produce. A variety of aptamers described to date have been shown to be reliable in modulating immune responses against cancer by either blocking or activating immune receptors. Some of them have been conjugated to other molecules to target the immune system and reduce off-target side effects. Despite the success of first-line treatments against cancer, the elevated number of relapsing cases and the tremendous side effects shown by the commonly used agents hinder conventional treatments against cancer. The advantages provided by aptamers could enhance the therapeutic index of a given strategy and therefore enhance the antitumor effect. Here we recapitulate the provided benefits of aptamers with immunomodulatory activity described to date in cancer therapy and the benefits that aptamer-based immunotherapy could provide either alone or combined with first-line treatments in cancer therapy.

  8. Aptamers: A Feasible Technology in Cancer Immunotherapy.

    Science.gov (United States)

    Soldevilla, M M; Villanueva, H; Pastor, F

    2016-01-01

    Aptamers are single-chained RNA or DNA oligonucleotides (ODNs) with three-dimensional folding structures which allow them to bind to their targets with high specificity. Aptamers normally show affinities comparable to or higher than that of antibodies. They are chemically synthesized and therefore less expensive to manufacture and produce. A variety of aptamers described to date have been shown to be reliable in modulating immune responses against cancer by either blocking or activating immune receptors. Some of them have been conjugated to other molecules to target the immune system and reduce off-target side effects. Despite the success of first-line treatments against cancer, the elevated number of relapsing cases and the tremendous side effects shown by the commonly used agents hinder conventional treatments against cancer. The advantages provided by aptamers could enhance the therapeutic index of a given strategy and therefore enhance the antitumor effect. Here we recapitulate the provided benefits of aptamers with immunomodulatory activity described to date in cancer therapy and the benefits that aptamer-based immunotherapy could provide either alone or combined with first-line treatments in cancer therapy.

  9. Function and dynamics of aptamers: A case study on the malachite green aptamer

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Tianjiao [Iowa State Univ., Ames, IA (United States)

    2008-01-01

    Aptamers are short single-stranded nucleic acids that can bind to their targets with high specificity and high affinity. To study aptamer function and dynamics, the malachite green aptamer was chosen as a model. Malachite green (MG) bleaching, in which an OH- attacks the central carbon (C1) of MG, was inhibited in the presence of the malachite green aptamer (MGA). The inhibition of MG bleaching by MGA could be reversed by an antisense oligonucleotide (AS) complementary to the MGA binding pocket. Computational cavity analysis of the NMR structure of the MGA-MG complex predicted that the OH- is sterically excluded from the C1 of MG. The prediction was confirmed experimentally using variants of the MGA with changes in the MG binding pocket. This work shows that molecular reactivity can be reversibly regulated by an aptamer-AS pair based on steric hindrance. In addition to demonstrate that aptamers could control molecular reactivity, aptamer dynamics was studied with a strategy combining molecular dynamics (MD) simulation and experimental verification. MD simulation predicted that the MG binding pocket of the MGA is largely pre-organized and that binding of MG involves reorganization of the pocket and a simultaneous twisting of the MGA terminal stems around the pocket. MD simulation also provided a 3D-structure model of unoccupied MGA that has not yet been obtained by biophysical measurements. These predictions were consistent with biochemical and biophysical measurements of the MGA-MG interaction including RNase I footprinting, melting curves, thermodynamic and kinetic constants measurement. This work shows that MD simulation can be used to extend our understanding of the dynamics of aptamer-target interaction which is not evident from static 3D-structures. To conclude, I have developed a novel concept to control molecular reactivity by an aptamer based on steric protection and a strategy to study the dynamics of aptamer-target interaction by combining MD

  10. High Efficiency Acetylcholinesterase Immobilization on DNA Aptamer Modified Surfaces

    Directory of Open Access Journals (Sweden)

    Orada Chumphukam

    2014-04-01

    Full Text Available We report here the in vitro selection of DNA aptamers for electric eel acetylcholinesterase (AChE. One selected aptamer sequence (R15/19 has a high affinity towards the enzyme (Kd = 157 ± 42 pM. Characterization of the aptamer showed its binding is not affected by low ionic strength (~20 mM, however significant reduction in affinity occurred at high ionic strength (~1.2 M. In addition, this aptamer does not inhibit the catalytic activity of AChE that we exploit through immobilization of the DNA on a streptavidin-coated surface. Subsequent immobilization of AChE by the aptamer results in a 4-fold higher catalytic activity when compared to adsorption directly on to plastic.

  11. Screening of aptamers on microfluidic systems for clinical applications.

    Science.gov (United States)

    Weng, Chen-Hsun; Huang, Chao-Jyun; Lee, Gwo-Bin

    2012-01-01

    The use of microfluidic systems for screening of aptamers and their biomedical applications are reviewed in this paper. Aptamers with different nucleic acid sequences have been extensively studied and the results demonstrated a strong binding affinity to target molecules such that they can be used as promising candidate biomarkers for diagnosis and therapeutics. Recently, the aptamer screening protocol has been conducted with microfluidic-based devices. Furthermore, aptamer affinity screening by a microfluidic-based method has demonstrated remarkable advantages over competing traditional methods. In this paper, we first reviewed microfluidic systems which demonstrated efficient and rapid screening of a specific aptamer. Then, the clinical applications of screened aptamers, also performed by microfluidic systems, are further reviewed. These automated microfluidic systems can provide advantages over their conventional counterparts including more compactness, faster analysis, less sample/reagent consumption and automation. An aptamer-based compact microfluidic system for diagnosis may even lead to a point-of-care device. The use of microfluidic systems for aptamer screening and diagnosis is expected to continue growing in the near future and may make a substantial impact on biomedical applications.

  12. Screening of Aptamers on Microfluidic Systems for Clinical Applications

    Directory of Open Access Journals (Sweden)

    Gwo-Bin Lee

    2012-07-01

    Full Text Available The use of microfluidic systems for screening of aptamers and their biomedical applications are reviewed in this paper. Aptamers with different nucleic acid sequences have been extensively studied and the results demonstrated a strong binding affinity to target molecules such that they can be used as promising candidate biomarkers for diagnosis and therapeutics. Recently, the aptamer screening protocol has been conducted with microfluidic-based devices. Furthermore, aptamer affinity screening by a microfluidic-based method has demonstrated remarkable advantages over competing traditional methods. In this paper, we first reviewed microfluidic systems which demonstrated efficient and rapid screening of a specific aptamer. Then, the clinical applications of screened aptamers, also performed by microfluidic systems, are further reviewed. These automated microfluidic systems can provide advantages over their conventional counterparts including more compactness, faster analysis, less sample/reagent consumption and automation. An aptamer-based compact microfluidic system for diagnosis may even lead to a point-of-care device. The use of microfluidic systems for aptamer screening and diagnosis is expected to continue growing in the near future and may make a substantial impact on biomedical applications.

  13. Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5

    Science.gov (United States)

    Yim, Sanggyu; Jeong, Yong-Joo

    2015-01-01

    Surface protein hemagglutinin (HA) mediates the binding of influenza virus to host cell receptors containing sialic acid, facilitating the entry of the virus into host cells. Therefore, the HA protein is regarded as a suitable target for the development of influenza virus detection devices. In this study, we isolated single-stranded DNA (ssDNA) aptamers binding to the HA1 subunit of subtype H1 (H1-HA1), but not to the HA1 subunit of subtype H5 (H5-HA1), using a counter-systematic evolution of ligands by exponential enrichment (counter-SELEX) procedure. Enzyme-linked immunosorbent assay and surface plasmon resonance studies showed that the selected aptamers bind tightly to H1-HA1 with dissociation constants in the nanomolar range. Western blot analysis demonstrated that the aptamers were binding to H1-HA1 in a concentration-dependent manner, yet were not binding to H5-HA1. Interestingly, the selected aptamers contained G-rich sequences in the central random nucleotides region. Further biophysical analysis showed that the G-rich sequences formed a G-quadruplex structure, which is a distinctive structure compared to the starting ssDNA library. Using flow cytometry analysis, we found that the aptamers did not bind to the receptor-binding site of H1-HA1. These results indicate that the selected aptamers that distinguish H1-HA1 from H5-HA1 can be developed as unique probes for the detection of the H1 subtype of influenza virus. PMID:25901739

  14. Aptamer-based nanobiosensors.

    Science.gov (United States)

    Kim, Yeon Seok; Raston, Nurul Hanun Ahmad; Gu, Man Bock

    2016-02-15

    It has been more than two decades since aptamer and the systematic evolution of ligands by exponential enrichment (SELEX) method were discovered by Larry Gold and Andrew Ellington in 1990, respectively. Based on the various advantages of aptamers, they have become a potent rival of antibodies in therapeutics and bio-analysis. Especially, the recent advances in aptamer biosensor application are remarkable due to its intrinsic properties of aptamers as nucleic acids and target induced conformational changes, in addition to the introduction of graphene oxide-based easy and simple immobilization-free screening method even for dual aptamers. In addition, the incorporation of various nanomaterials such as metallic nanoparticles, carbon materials, and functional nanospheres in aptasensors has facilitated the improvement of analytical performance and commercial application of aptasensors. In this review, recent prominent reports on aptasensors utilizing nanomaterials were introduced to understand the principle of aptamer-based biosensors and provide an insight for new strategies of aptasensors and the application of various nanomaterials. The perspective on aptamer-based biosensors and diagnostics was also discussed in view of technology and market.

  15. Aptamer-Functionalized Nano-Biosensors

    Directory of Open Access Journals (Sweden)

    Tai-Chia Chiu

    2009-12-01

    Full Text Available Nanomaterials have become one of the most interesting sensing materials because of their unique size- and shape-dependent optical properties, high surface energy and surface-to-volume ratio, and tunable surface properties. Aptamers are oligonucleotides that can bind their target ligands with high affinity. The use of nanomaterials that are bioconjugated with aptamers for selective and sensitive detection of analytes such as small molecules, metal ions, proteins, and cells has been demonstrated. This review focuses on recent progress in the development of biosensors by integrating functional aptamers with different types of nanomaterials, including quantum dots, magnetic nanoparticles (NPs, metallic NPs, and carbon nanotubes. Colorimetry, fluorescence, electrochemistry, surface plasmon resonance, surface-enhanced Raman scattering, and magnetic resonance imaging are common detection modes for a broad range of analytes with high sensitivity and selectivity when using aptamer bioconjugated nanomaterials (Apt-NMs. We highlight the important roles that the size and concentration of nanomaterials, the secondary structure and density of aptamers, and the multivalent interactions play in determining the specificity and sensitivity of the nanosensors towards analytes. Advantages and disadvantages of the Apt-NMs for bioapplications are focused.

  16. High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase.

    Science.gov (United States)

    Ditzler, Mark A; Lange, Margaret J; Bose, Debojit; Bottoms, Christopher A; Virkler, Katherine F; Sawyer, Andrew W; Whatley, Angela S; Spollen, William; Givan, Scott A; Burke, Donald H

    2013-02-01

    Systematic evolution of ligands through exponential enrichment (SELEX) is a well-established method for generating nucleic acid populations that are enriched for specified functions. High-throughput sequencing (HTS) enhances the power of comparative sequence analysis to reveal details of how RNAs within these populations recognize their targets. We used HTS analysis to evaluate RNA populations selected to bind type I human immunodeficiency virus reverse transcriptase (RT). The populations are enriched in RNAs of independent lineages that converge on shared motifs and in clusters of RNAs with nearly identical sequences that share common ancestry. Both of these features informed inferences of the secondary structures of enriched RNAs, their minimal structural requirements and their stabilities in RT-aptamer complexes. Monitoring population dynamics in response to increasing selection pressure revealed RNA inhibitors of RT that are more potent than the previously identified pseudoknots. Improved potency was observed for inhibition of both purified RT in enzymatic assays and viral replication in cell-based assays. Structural and functional details of converged motifs that are obscured by simple consensus descriptions are also revealed by the HTS analysis. The approach presented here can readily be generalized for the efficient and systematic post-SELEX development of aptamers for down-stream applications.

  17. Aptamer-targeted DNA nanostructures for therapeutic delivery.

    Science.gov (United States)

    Charoenphol, Phapanin; Bermudez, Harry

    2014-05-05

    DNA-based nanostructures have been widely used in various applications due to their structural diversity, programmability, and uniform structures. Their intrinsic biocompatibility and biodegradability further motivates the investigation of DNA-based nanostructures as delivery vehicles. Incorporating AS1411 aptamers into DNA pyramids leads to enhanced intracellular uptake and selectively inhibits the growth of cancer cells, achieved without the use of transfection reagents. Furthermore, aptamer-displaying pyramids are found to be substantially more resistant to nuclease degradation than single-stranded aptamers. These findings, along with their modularity, reinforce the potential of DNA-based nanostructures for therapeutic applications.

  18. Fibonacci Sequence and Supramolecular Structure of DNA.

    Science.gov (United States)

    Shabalkin, I P; Grigor'eva, E Yu; Gudkova, M V; Shabalkin, P I

    2016-05-01

    We proposed a new model of supramolecular DNA structure. Similar to the previously developed by us model of primary DNA structure [11-15], 3D structure of DNA molecule is assembled in accordance to a mathematic rule known as Fibonacci sequence. Unlike primary DNA structure, supramolecular 3D structure is assembled from complex moieties including a regular tetrahedron and a regular octahedron consisting of monomers, elements of the primary DNA structure. The moieties of the supramolecular DNA structure forming fragments of regular spatial lattice are bound via linker (joint) sequences of the DNA chain. The lattice perceives and transmits information signals over a considerable distance without acoustic aberrations. Linker sequences expand conformational space between lattice segments allowing their sliding relative to each other under the action of external forces. In this case, sliding is provided by stretching of the stacked linker sequences.

  19. Novel protein detection method based on proximity-dependent polymerase reaction and aptamers

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In recent years, specific detection of proteins is one of the hot issues about aptamers in proteomics.Here we reported a simple, sensitive and specific proximity-dependent protein assay with dual DNA aptamers. Thrombin was used as the model protein, and two aptamer probes with complementary sequence at 3'-end were designed for the two distinct epitopes of the protein. Association of the two aptamers with thrombin resulted in stable hybrids due to the proximity of 3'-end, then polymerase reaction was induced. The amount of obtained dsDNA was indicated using the fluorescence dye Sybr Green 1. The results showed that the initial velocity of polymerase reaction had a positive correlation with concentration of thrombin. The advantages of this dual-aptamer-based approach included simple and flexible design of aptamer probes, high selectivity and high sensitivity. The detection limit was 6.9pmol/L.

  20. Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against alpha-Bungarotoxin

    DEFF Research Database (Denmark)

    Lauridsen, Lasse Holm; Shamaileh, Hadi A.; Edwards, Stacey L.

    2012-01-01

    Background: Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen (R), an inhibitor of vascular endothelial growth factor (VEGF...... by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 mu M. Conclusion: We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low...... micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way...

  1. Aptamer modification improves the adenoviral transduction of malignant glioma cells.

    Science.gov (United States)

    Chen, Hao; Zheng, Xiaojing; Di, BingYan; Wang, Dongyang; Zhang, Yaling; Xia, Haibin; Mao, Qinwen

    2013-12-01

    Adenovirus has shown increasing promise in the gene-viral therapy for glioblastoma, a treatment strategy that relies on the delivery of viruses or transgenes into tumor cells. However, targeting of adenovirus to human glioblastoma remains a challenge due to the low expression level of coxsackie and adenovirus receptor (CAR) in glioma cells. Aptamers are small and highly structured single-stranded oligonucleotides that bind at high affinity to a target molecule, and are good candidates for targeted imaging and therapy. In this study, to construct an aptamer-modified Ad5, we first genetically modified the HVR5 of Ad hexon by biotin acceptor peptide (BAP), which would be metabolically biotinylated during production in HEK293 cells, and then attached the biotin labeled aptamer to the modified Ad through avidin–biotin binding. The aptamers used in this study includes AS1411 and GBI-10. The former is a DNA aptamer that can bind to nucleolin, a nuclear matrix protein found on the surface of cancer cells. The latter is a DNA aptamer that can recognize the extracellular matrix protein tenascin-C on the surface of human glioblastoma cells. To examine if aptamer-modification of the hexon protein could improve the adenoviral transduction efficiency, a glioblastoma cell line, U251, was transduced with aptamer-modified Ads. The transduction efficiency of AS1411- or GBI-10-modified Ad was approximately 4.1-fold or 5.2-fold higher than that of the control. The data indicated that aptamer modified adenovirus would be a useful tool for cancer gene therapy.

  2. Development of aptamers for use as radiopharmaceuticals in the bacterial infection identification; Desenvolvimento de aptameros especificos para aplicacao como radiofarmacos na identificacao de bacterias

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Ieda Mendes

    2013-08-01

    were cloned. The product of recombination was used to transform Escherichia coli ToplOF'. The plasmid DNA from 40 selected colonies were extracted and quantified. The plasmids were sequenced, two different sequences (Antibac1 and Antibac2) were obtained and their secondary structures determined. The aptamers obtained were synthesized and labeled with {sup 32}p. The labeled aptamers were incubated with S. aureus cells and the amount of radiolabeled ssDNA was determined by liquid scintillation spectrometry. The oligonucleotides library labeled with {sup 32}p was used as control. For the aptamer Antibac1 the radiation linked to the pellet was 28 times higher than obtained with the control and for the Antibac2 was 22 higher. A specific assay was conducted with labeled aptamers using S. aureus, E. co ti, Candida albicans and human fibroblasts. For both aptamers (Antibac1 and Antibac2) binding to bacterial cells was significantly higher than for C. albicans and fibroblasts, demonstrating their specificity for bacteria. For B process, after 15 SELEX rounds was performed the cell-SELEX starting with the products 15th round being incubated with S. aureus cells. The amplified oligonucleotides were incubated again with the bacteria in the next round and so on. At the end of the 5 rounds of selection, the selected oligonucleotides were cloned and sequenced as in A process. Eleven different sequences from 21 clones were obtained and their secondary structures were determined. The aptamers obtained by process A showed high affinity and specificity for bacteria. The aptamers obtained by process B will be evaluated for these parameters in a future work. (author)

  3. Modeling the microscopic electrical properties of thrombin binding aptamer (TBA) for label-free biosensors

    CERN Document Server

    Alfinito, Eleonora; Cataldo, Rosella; De Nunzio, Giorgio; Giotta, Livia; Guascito, Maria Rachele

    2016-01-01

    Aptamers are chemically produced oligonucleotides, able to bind a variety of targets such as drugs, proteins and pathogens with high sensitivity and selectivity. Therefore, aptamers are largely employed for producing label-free biosensors, with significant applications in diagnostics and drug delivery. In particular, the anti-thrombin aptamers are biomolecules of high interest for clinical use, because of their ability to recognize and bind the thrombin enzyme. Among them, the DNA 15-mer thrombin-binding aptamer (TBA), has been widely explored concerning both its structure, which was resolved with different techniques, and its function, especially about the possibility of using it as the active part of biosensors. This paper proposes a microscopic model of the electrical properties of TBA and the aptamer-thrombin complex, combining information from both structure and function. The novelty consists in describing both the aptamer alone and the complex as an impedance network, thus going deeper inside the issues...

  4. Recent advances of aptamer sensors

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Aptamers are a series of high-affinity and high-specificity oligoneucleotides (single-stranded DNA or RNA) to the target, usually selected by the combinatorial chemistry SELEX technique (systematic evolution of ligands by exponential enrichment). Aptamers have proved to be one kind of novel functional molecules in life science and chemistry. After being labeled by signaling groups, the aptamer probe can conveniently transfer the characteristics of aptamer-target recognition to a form of high-sensitive signal, and the high-affinity, high-specificity measurements of metal ion, organic molecules, nucleic acid, proteins, or cells become possible. This article summarizes the recent advances of aptamer probes in different sensing fields, with special emphasis on aptamer probes as fluorescent sensors.

  5. Recent advances of aptamer sensors

    Institute of Scientific and Technical Information of China (English)

    LI YiLin; GUO Lei; ZHANG ZhaoYang; TANG JiJun; XIE JianWei

    2008-01-01

    Aptamers are a series of high-affinity and high-specificity oligoneucleotides (single-stranded DNA or RNA) to the target, usually selected by the combinatorial chemistry SELEX technique (systematic evolution of ligands by exponential enrichment). Aptamers have proved to be one kind of novel func-tional molecules in life science and chemistry. After being labeled by signaling groups, the aptamer probe can conveniently transfer the characteristics of aptamer-target recognition to a form of high-sensitive signal, and the high-affinity, high-specificity measurements of metal ion, organic mole-cules, nucleic acid, proteins, or cells become possible. This article summarizes the recent advances of aptamer probes in different sensing fields, with special emphasis on aptamer probes as fluorescent sensors.

  6. Potent Inhibition of HIV-1 Reverse Transcriptase and Replication by Nonpseudoknot, "UCAA-motif" RNA Aptamers.

    Science.gov (United States)

    Whatley, Angela S; Ditzler, Mark A; Lange, Margaret J; Biondi, Elisa; Sawyer, Andrew W; Chang, Jonathan L; Franken, Joshua D; Burke, Donald H

    2013-02-05

    RNA aptamers that bind the reverse transcriptase (RT) of human immunodeficiency virus (HIV) compete with nucleic acid primer/template for access to RT, inhibit RT enzymatic activity in vitro, and suppress viral replication when expressed in human cells. Numerous pseudoknot aptamers have been identified by sequence analysis, but relatively few have been confirmed experimentally. In this work, a screen of nearly 100 full-length and >60 truncated aptamer transcripts established the predictive value of the F1Pk and F2Pk pseudoknot signature motifs. The screen also identified a new, nonpseudoknot motif with a conserved unpaired UCAA element. High-throughput sequence (HTS) analysis identified 181 clusters capable of forming this novel element. Comparative sequence analysis, enzymatic probing and RT inhibition by aptamer variants established the essential requirements of the motif, which include two conserved base pairs (AC/GU) on the 5' side of the unpaired UCAA. Aptamers in this family inhibit RT in primer extension assays with IC(50) values in the low nmol/l range, and they suppress viral replication with a potency that is comparable with that of previously studied aptamers. All three known anti-RT aptamer families (pseudoknots, the UCAA element, and the recently described "(6/5)AL" motif) are therefore suitable for developing aptamer-based antiviral gene therapies.Molecular Therapy - Nucleic Acids (2013) 2, e71; doi:10.1038/mtna.2012.62; published online 5 February 2013.

  7. Use of aptamers in immunoassays.

    Science.gov (United States)

    Nezlin, Roald

    2016-02-01

    Aptamers, short single-chain DNA or RNA oligonucleotides, react specifically with small molecules, as well as with proteins. Unlike antibodies, they may be obtained relatively easily. Aptamers are now widely employed in immunological studies and could replace antibodies in immunoassays. In this short review, methods for immobilizing aptamers on various insoluble materials (so-called apta-sorbents) are described. Recent findings on their use in the detection and isolation of immunoglobulins and their application in various immunoassays are also discussed.

  8. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

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

    2011-01-01

    Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing...... strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers...

  9. Modeling the microscopic electrical properties of thrombin binding aptamer (TBA) for label-free biosensors

    Science.gov (United States)

    Alfinito, Eleonora; Reggiani, Lino; Cataldo, Rosella; De Nunzio, Giorgio; Giotta, Livia; Guascito, Maria Rachele

    2017-02-01

    Aptamers are chemically produced oligonucleotides, able to bind a variety of targets such as drugs, proteins and pathogens with high sensitivity and selectivity. Therefore, aptamers are largely employed for producing label-free biosensors (aptasensors), with significant applications in diagnostics and drug delivery. In particular, the anti-thrombin aptamers are biomolecules of high interest for clinical use, because of their ability to recognize and bind the thrombin enzyme. Among them, the DNA 15-mer aptamer (TBA), has been widely explored around the possibility of using it in aptasensors. This paper proposes a microscopic model of the electrical properties of TBA and of the aptamer-thrombin complex, combining information from both structure and function, following the issues addressed in an emerging branch of electronics known as proteotronics. The theoretical results are compared and validated with measurements reported in the literature. Finally, the model suggests resistance measurements as a novel tool for testing aptamer-target affinity.

  10. IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION

    Science.gov (United States)

    Boese, B. J.; Corbino, K.; Breaker, R. R.

    2017-01-01

    We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust affinity for cellulose in both the powdered and paper form, but did not show any significant affinity for closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using the glucosamine 6-phosphate to activate glmS ribozyme function. PMID:18696364

  11. Aptamer-gelatin composite for a trigger release system mediated by oligonucleotide hybridization.

    Science.gov (United States)

    Soontornworajit, Boonchoy; Srakaew, Prangkamol; Naramitpanich, Pajaree

    2014-01-01

    Nucleic acid aptamers not only specifically bind to their target proteins with high affinity but also form intermolecular hybridization with their complementary oligonucleotides (CO). The hybridization can interrupt aptamer/protein interaction due to the changes of aptamer secondary structure which rely on hybridization length and base-pairing positions. Herein we aim to use this unique property of the aptamers, when combined with gelatin to develop a novel composite with desirable protein release profiles. Platelet-derived growth factor-BB (PDGF-BB) and its aptamer were used as target molecules. Prior to performing the release study, the effects of CO on aptamer-protein interaction were observed by surface plasmon resonance (SPR). The SPR sensorgram indicated that the aptamer dissociated from the bounded proteins when it hybridized with the CO. The aptamer was then immobilized onto streptavidin coated polystyrene particles via biotin/streptavidin interaction. Then, PDGF-BB and aptamer functionalized particles were mixed with gelatin solution and cast as small pieces of composite. The success of the composite preparation was confirmed by flow cytometry and microscopy. PDGF-BB release at several time points was quantified by ELISA. The results showed that the aptamer-gelatin composite could slow the release rate of the proteins from the composite due to strong binding of proteins and aptamers. Once the CO was added to the system, the release rate was significantly enhanced because the aptamer hybridized with the CO and lost its active secondary structure. Therefore, the proteins were triggered to release out from the composite. This work suggests a promising strategy for controlling the release of bioactive molecules in medical treatments.

  12. Selection of LNA-containing DNA aptamers against recombinant human CD73.

    Science.gov (United States)

    Elle, Ida C; Karlsen, Kasper K; Terp, Mikkel G; Larsen, Niels; Nielsen, Ronni; Derbyshire, Nicola; Mandrup, Susanne; Ditzel, Henrik J; Wengel, Jesper

    2015-05-01

    LNA-containing DNA aptamers against CD73 (human ecto-5'-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several steps including sequence unification, clustering and alignment to identify enriched sequences. Three enriched sequences were synthesised for further analysis, two of which showed sequence similarities. These sequences exhibited binding to the recombinant CD73 with KD values of 10 nM and 3.5 nM when tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences were able to decrease the activity of the protein. However, the aptamers exhibited no binding to cellular CD73 by flow cytometry analysis likely since the epitope recognised by the aptamer was not available for binding on the cellular protein.

  13. Aptamer-Drug Conjugates.

    Science.gov (United States)

    Zhu, Guizhi; Niu, Gang; Chen, Xiaoyuan

    2015-11-18

    Western medicine often aims to specifically treat diseased tissues or organs. However, the majority of current therapeutics failed to do so owing to their limited selectivity and the consequent "off-target" side effects. Targeted therapy aims to enhance the selectivity of therapeutic effects and reduce adverse side effects. One approach toward this goal is to utilize disease-specific ligands to guide the delivery of less-specific therapeutics, such that the therapeutic effects can be guided specifically to diseased tissues or organs. Among these ligands, aptamers, also known as chemical antibodies, have emerged over the past decades as a novel class of targeting ligands that are capable of specific binding to disease biomarkers. Compared with other types of targeting ligands, aptamers have an array of unique advantageous features, which make them promising for developing aptamer-drug conjugates (ApDCs) for targeted therapy. In this Review, we will discuss ApDCs for targeted drug delivery in chemotherapy, gene therapy, immunotherapy, photodynamic therapy, and photothermal therapy, primarily of cancer.

  14. Ligand Binding and Conformational Changes in the Purine-Binding Riboswitch Aptamer Domains

    Science.gov (United States)

    Noeske, Jonas; Buck, Janina; Wöhnert, Jens; Schwalbe, Harald

    Riboswitches are highly structured mRNA elements that regulate gene expression upon specific binding of small metabolite molecules. The purine-binding riboswitches bind different purine ligands by forming both canonical Watson—Crick and non-canonical intermolecular base pairs, involving a variety of hydrogen bonds between the riboswitch aptamer domain and the purine ligand. Here, we summarize work on the ligand binding modes of both purine-binding aptamer domains, their con-formational characteristics in the free and ligand-bound forms, and their ligand-induced folding. The adenine- and guanine-binding riboswitch aptamer domains display different conformations in their free forms, despite nearly identical nucleotide loop sequences that form a loop—loop interaction in the ligand-bound forms. Interestingly, the stability of helix II is crucial for the formation of the loop—loop interaction in the free form. A more stable helix II in the guanine riboswitch leads to a preformed loop—loop interaction in its free form. In contrast, a less stable helix II in the adenine riboswitch results in a lack of this loop—loop interaction in the absence of ligand and divalent cations.

  15. Robust aptamer sol-gel solid phase microextraction of very polar adenosine from human plasma.

    Science.gov (United States)

    Mu, Li; Hu, Xiangang; Wen, Jianping; Zhou, Qixing

    2013-03-01

    Conventional solid phase microextraction (SPME) has a limited capacity to extract very polar analytes, such as adenosine. To solve this problem, aptamer conjugating sol-gel methodology was coupled with an SPME fiber. According to the authors' knowledge, this is the first reported use of aptamer SPME. The fiber of aptamer sol-gel SPME with a mesoporous structure has high porosity, large surface area, and small water contact angle. Rather than employing direct entrapment, covalent immobilization was the dominant method of aptamer loading in sol-gel. Aptamer sol-gel fiber captured a specified analyte from among the analog molecules, thereby, exhibiting an excellent selective property. Compared with commercial SPME fibers, this aptamer fiber was suitable for extracting adenosine, presenting an extraction efficiency higher than 20-fold. The values of repeatability and reproducibility expressed by relative standard deviation were low (9.4%). Interestingly, the sol-gel network enhanced the resistance of aptamer SPME to both nuclease and nonspecific proteins. Furthermore, the aptamer sol-gel fiber was applied in human plasma with LOQ 1.5 μg/L, which is an acceptable level. This fiber also demonstrates durability and regeneration over 20-cycles without significant loss of efficiency. Given the various targets (from metal ions to biomacromolecules and cells) of aptamers, this methodology will extend the multi-domain applications of SPME.

  16. Inhibition of HCV NS3 protease by RNA aptamers in cells.

    Science.gov (United States)

    Nishikawa, Fumiko; Kakiuchi, Nobuko; Funaji, Kohei; Fukuda, Kotaro; Sekiya, Satoru; Nishikawa, Satoshi

    2003-04-01

    Non-structural protein 3 (NS3) of hepatitis C virus (HCV) has two distinct activities, protease and helicase, which are essential for HCV proliferation. In previous work, we obtained RNA aptamers (G9-I, II and III) which specifically bound the NS3 protease domain (DeltaNS3), efficiently inhibiting protease activity in vitro. To utilize these aptamers in vivo, we constructed a G9 aptamer expression system in cultured cells, using the cytomegarovirus enhancer + chicken beta-actin globin (CAG) promoter. By conjugating the cis-acting genomic human hepatitis delta virus (HDV) ribozyme and G9-II aptamer, a chimeric HDV ribozyme-G9-II aptamer (HA) was constructed, which was used to produce stable RNA in vivo and to create tandem repeats of the functional unit. To target the transcribed RNA aptamers to the cytoplasm, the minimal mutant of constitutive transport element (CTE), derived from type D retroviruses, was conjugated at the 3' end of HA (HAC). Transcript RNAs from (HA)(n) and (HAC)(n) were processed into the G9-II aptamer unit by the cis-acting HDV ribozyme, both in vitro and in vivo. Efficient protease inhibition activity of HDV ribozyme-G9-II aptamer expression plasmid was demonstrated in HeLa cells. Protease inhibition activity level of tandem chimeric aptamers, (HA)(n) and (HAC)(n), rose with the increase of n from 1 to 4.

  17. Immuno Affinity SELEX for Simple, Rapid, and Cost-Effective Aptamer Enrichment and Identification against Aflatoxin B1

    Science.gov (United States)

    Setlem, Keerthana; Mondal, Bhairab; Ramlal, Shylaja; Kingston, Joseph

    2016-01-01

    Aflatoxins are naturally occurring mycotoxins that contaminate food and agro commodities, leading to acute and chronic health conditions in human and animals. In the present work, an attempt was made to generate high-affinity single stranded DNA aptamers that specifically bind to Aflatoxin B1 (AFB1) by a modified Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure with the aid of Immunoaffinity columns. Ten rounds of SELEX and alternating three counter SELEX rounds with a cocktail of related and other mycotoxins were performed to enhance the specificity. Resultant 105 aptamers were clustered into 12 groups according to their primary sequence homology. Candidates with lowest Gibbs free energy (dG value) and unique stem loop structures were selected for further characterization. Aptamers, AFLA5, AFLA53, and AFLA71 exhibiting lower Kd values (50.45 ± 11.06, 48.29 ± 9.45, and 85.02 ± 25.74 nM) were chosen for development of ELONA and determination of purification ability of toxin. The detection limit (LOD) of AFLA5 and AFLA71 was 20 and 40 ng/ml, respectively. HPLC analysis implied that selected aptamers were able to recover and quantify 82.2 to 96.21% (LOQ – 53.74 ng) and 78.3 to 94.22% (LOQ – 66.75 ng) of AFB1 from spiked corn samples, respectively. These findings indicate, immunoaffinity based SELEX can pave an alternative approach to screen aptamers against mycotoxin detection and purification. PMID:27990137

  18. Protein Detection with Aptamer Biosensors

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    2008-07-01

    Full Text Available Aptamers have been developed for different applications. Their use as new biological recognition elements in biosensors promises progress for fast and easy detection of proteins. This new generation of biosensor (aptasensors will be more stable and well adapted to the conditions of real samples because of the specific properties of aptamers.

  19. Computational Selection of RNA Aptamer against Angiopoietin-2 and Experimental Evaluation

    Directory of Open Access Journals (Sweden)

    Wen-Pin Hu

    2015-01-01

    Full Text Available Angiogenesis plays a decisive role in the growth and spread of cancer and angiopoietin-2 (Ang2 is in the spotlight of studies for its unique role in modulating angiogenesis. The aim of this study was to introduce a computational simulation approach to screen aptamers with high binding ability for Ang2. We carried out computational simulations of aptamer-protein interactions by using ZDOCK and ZRANK functions in Discovery Studio 3.5 starting from the available information of aptamers generated through the systematic evolution of ligands by exponential enrichment (SELEX in the literature. From the best of three aptamers on the basis of ZRANK scores, 189 sequences with two-point mutations were created and simulated with Ang2. Then, we used a surface plasmon resonance (SPR biosensor to test 3 mutant sequences of high ZRANK scores along with a high and a low affinity binding sequence as reported in the literature. We found a selected RNA aptamer has a higher binding affinity and SPR response than a reported sequence with the highest affinity. This is the first study of in silico selection of aptamers against Ang2 by using the ZRANK scoring function, which should help to increase the efficiency of selecting aptamers with high target-binding ability.

  20. Selection of LNA-containing DNA aptamers against recombinant human CD73

    DEFF Research Database (Denmark)

    Elle, Ida C; Karlsen, Kasper K; Terp, Mikkel G

    2015-01-01

    tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences......LNA-containing DNA aptamers against CD73 (human ecto-5'-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX...... with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several...

  1. Selective Aptamers for Detection of Estradiol and Ethynylestradiol in Natural Waters

    KAUST Repository

    Akki, Spurti U.

    2015-08-18

    © 2015 American Chemical Society. We used in vitro selection to identify new DNA aptamers for two endocrine-disrupting compounds often found in treated and natural waters, 17β-estradiol (E2) and 17α-ethynylestradiol (EE). We used equilibrium filtration to determine aptamer sensitivity/selectivity and dimethyl sulfate (DMS) probing to explore aptamer binding sites. The new E2 aptamers are at least 74-fold more sensitive for E2 than is a previously reported DNA aptamer, with dissociation constants (Kd values) of 0.6 μM. Similarly, the EE aptamers are highly sensitive for EE, with Kd of 0.5-1.0 μM. Selectivity values indicate that the E2 aptamers bind E2 and a structural analogue, estrone (E1), equally well and are up to 74-fold selective over EE. One EE aptamer is 53-fold more selective for EE over E2 or E1, but the other binds EE, E2, and E1 with similar affinity. The new aptamers do not lose sensitivity or selectivity in natural water from a local lake, despite the presence of natural organic matter (∼4 mg/L TOC). DMS probing suggests that E2 binding occurs in relatively flexible single-stranded DNA regions, an important finding for rational redesign of aptamers and their incorporation into sensing platforms. This is the first report of aptamers with strong selectivity for E2 and E1 over EE, or with strong selectivity for EE over E2 and E1. Such selectivity is important for achieving the goal of creating practically useful DNA-based sensors that can distinguish structurally similar estrogenic compounds in natural waters.

  2. Riboswitch Structure: an Internal Residue Mimicking the Purine Ligand

    Energy Technology Data Exchange (ETDEWEB)

    Delfosse, V.; Bouchard, P; Bonneau, E; Dagenais, P; Lemay, J; Lafontaine, D; Legault, P

    2009-01-01

    The adenine and guanine riboswitches regulate gene expression in response to their purine ligand. X-ray structures of the aptamer moiety of these riboswitches are characterized by a compact fold in which the ligand forms a Watson-Crick base pair with residue 65. Phylogenetic analyses revealed a strict restriction at position 39 of the aptamer that prevents the G39-C65 and A39-U65 combinations, and mutational studies indicate that aptamers with these sequence combinations are impaired for ligand binding. In order to investigate the rationale for sequence conservation at residue 39, structural characterization of the U65C mutant from Bacillus subtilis pbuE adenine riboswitch aptamer was undertaken. NMR spectroscopy and X-ray crystallography studies demonstrate that the U65C mutant adopts a compact ligand-free structure, in which G39 occupies the ligand-binding site of purine riboswitch aptamers. These studies present a remarkable example of a mutant RNA aptamer that adopts a native-like fold by means of ligand mimicking and explain why this mutant is impaired for ligand binding. Furthermore, this work provides a specific insight into how the natural sequence has evolved through selection of nucleotide identities that contribute to formation of the ligand-bound state, but ensures that the ligand-free state remains in an active conformation.

  3. Selection and Characterization of a Novel DNA Aptamer for Label-Free Fluorescence Biosensing of Ochratoxin A

    Directory of Open Access Journals (Sweden)

    Maureen McKeague

    2014-08-01

    Full Text Available Nucleic acid aptamers are emerging as useful molecular recognition tools for food safety monitoring. However, practical and technical challenges limit the number and diversity of available aptamer probes that can be incorporated into novel sensing schemes. This work describes the selection of novel DNA aptamers that bind to the important food contaminant ochratoxin A (OTA. Following 15 rounds of in vitro selection, sequences were analyzed for OTA binding. Two of the isolated aptamers demonstrated high affinity binding and selectivity to this mycotoxin compared to similar food adulterants. These sequences, as well as a truncated aptamer (minimal sequence required for binding, were incorporated into a SYBR® Green I fluorescence-based OTA biosensing scheme. This label-free detection platform is capable of rapid, selective, and sensitive OTA quantification with a limit of detection of 9 nM and linear quantification up to 100 nM.

  4. Discovery and development of anticancer aptamers.

    Science.gov (United States)

    Ireson, Christopher R; Kelland, Lloyd R

    2006-12-01

    Aptamers, also termed as decoys or "chemical antibodies," represent an emerging class of therapeutics. They are short DNA or RNA oligonucleotides or peptides that assume a specific and stable three-dimensional shape in vivo, thereby providing specific tight binding to protein targets. In some cases and as opposed to antisense oligonucleotides, effects can be mediated against extracellular targets, thereby preventing a need for intracellular transportation. The first aptamer approved for use in man is a RNA-based molecule (Macugen, pegaptanib) that is administered locally (intravitreally) to treat age-related macular degeneration by targeting vascular endothelial growth factor. The most advanced aptamer in the cancer setting is AS1411, formerly known as AGRO100, which is being administered systemically in clinical trials. AS1411 is a 26-mer unmodified guanosine-rich oligonucleotide, which induces growth inhibition in vitro, and has shown activity against human tumor xenografts in vivo. The mechanism underlying its antiproliferative effects in cancer cells seems to involve initial binding to cell surface nucleolin and internalization, leading to an inhibition of DNA replication. In contrast to other unmodified oligonucleotides, AS1411 is relatively stable in serum-containing medium, probably as a result of the formation of dimers and a quartet structure. In a dose escalation phase I study in patients with advanced solid tumors, doses up to 10 mg/kg/d (using a four or seven continuous infusion regime) have been studied. Promising signs of activity have been reported (multiple cases of stable disease and one near complete response in a patient with renal cancer) in the absence of any significant adverse effects. Further trials are ongoing in renal and non-small cell lung cancers. In preclinical studies, additional aptamers have been described against several cancer targets, such as tenascin-C, the transcription factor signal transducer and activator of transcription 3

  5. Aptamer-based surface plasmon resonance sensing of glycated human blood proteins

    Science.gov (United States)

    Reaver, Nathan G. F.; Zheng, Rui; Kim, Dong-Shik; Cameron, Brent D.

    2013-02-01

    The concentration ratio of glycated to non-glycated forms of various blood proteins can be used as a diagnostic measure in diabetes to determine a history of glycemic compliance. Depending on a protein's half-life in blood, compliance can be assessed from a few days to several months in the past, which can then be used to provide additional therapeutic guidance. Current glycated protein detection methods are limited in their ability to measure multiple proteins, and are susceptible to interference from other blood pathologies. In this study, we developed and characterized DNA aptamers for use in Surface Plasmon Resonance (SPR) sensors to assess the blood protein hemoglobin. The aptamers were developed by way of a modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process which selects DNA sequences that have a high binding affinity to a specific protein. DNA products resulting from this process are sequenced and identified aptamers are then synthesized. The SELEX process was performed to produce aptamers for a glycated form of hemoglobin. Equilibrium dissociation constants for the binding of the identified aptamer to glycated hemoglobin, hemoglobin, and fibrinogen were calculated from fitted Langmuir isotherms obtained through SPR. These constants were determined to be 94 nM, 147 nM, and 244 nM respectively. This aptamer can potentially be used to create a SPR aptamer based biosensor for detection of glycated hemoglobin, a technology that has the potential to deliver low-cost and immediate glycemic compliance assessment in either a clinical or home setting.

  6. A symmetry-related sequence-structure relation of proteins

    Institute of Scientific and Technical Information of China (English)

    XU Ruizhen; LI Mingfen; CHEN Hanlin; HUANG Yanzhao; XIAO Yi

    2005-01-01

    Proteins have regular tertiary structures but irregular amino acid sequences. This made it very difficult to decode the structural information in the protein sequences. Here we demonstrate that many small αprotein domains have hidden sequence symmetries characteristic of their pseudo-symmetric tertiary structures. We also present a modified method of recurrent plot to reveal this kind of the hidden sequence symmetry. The results may enable us to understand part of the relations between protein sequences and their tertiary structures.

  7. DNA nanostructure-decorated surfaces for enhanced aptamer-target binding and electrochemical cocaine sensors.

    Science.gov (United States)

    Wen, Yanli; Pei, Hao; Wan, Ying; Su, Yan; Huang, Qing; Song, Shiping; Fan, Chunhai

    2011-10-01

    The sensitivity of aptamer-based electrochemical sensors is often limited by restricted target accessibility and surface-induced perturbation of the aptamer structure, which arise from imperfect packing of probes on the heterogeneous and locally crowded surface. In this study, we have developed an ultrasensitive and highly selective electrochemical aptamer-based cocaine sensor (EACS), based on a DNA nanotechnology-based sensing platform. We have found that the electrode surface decorated with an aptamer probe-pendant tetrahedral DNA nanostructure greatly facilitates cocaine-induced fusion of the split anticocaine aptamer. This novel design leads to a sensitive cocaine sensor with a remarkably low detection limit of 33 nM. It is also important that the tetrahedra-decorated surface is protein-resistant, which not only suits the enzyme-based signal amplification scheme employed in this work, but ensures high selectivity of this sensor when deployed in sera or other adulterated samples.

  8. Glutamate Receptor Aptamers and ALS

    Science.gov (United States)

    2009-01-01

    proposed, including oxidative stress, excitotoxicity, mitochondrial dysfunction, etc., the cause(s) of the disease, including the pathogenesis of the...GluR6-Selective Aptamers for Potential Autism Therapy This project is to develop RNA aptamers against a GluR6 kainate receptor mutant thought to be...involved in autism . Role: PI Department of Defense (PI: Niu) 4/1/09-3/30/14 Advanced Tech./Therapeutic Develop. Grant Developing Biostable

  9. Development of a Sphingosylphosphorylcholine Detection System Using RNA Aptamers

    Directory of Open Access Journals (Sweden)

    Iwao Waga

    2010-08-01

    Full Text Available Sphingosylphosphorylcholine (SPC is a lysosphingolipid that exerts multiple functions, including acting as a spasmogen, as a mitogenic factor for various types of cells, and sometimes as an inflammatory mediator. Currently, liquid chromatography/tandem mass spectrometry (LC/MS/MS is used for the quantitation of SPC. However, because of the complicated procedures required it may not be cost effective, hampering its regular usage in a routine practical SPC monitoring. In this report, we have generated RNA aptamers that bind to SPC with high affinity using an in vitro selection procedure and developed an enzyme-linked aptamer assay system using the minimized SPC aptamer that can successfully distinguish SPC from the structurally related sphingosine 1-phosphate (S1P. This is the first case of the Systematic Evolution of Ligands by EXponential enrichment (SELEX process being performed with a lysosphingolipid. The SPC aptamers would be valuable tools for the development of aptamer-based medical diagnosis and for elucidating the biological role of SPC.

  10. Aptamers in Virology: Recent Advances and Challenges

    OpenAIRE

    Binning, Jennifer M.; Leung, Daisy W.; Amarasinghe, Gaya K.

    2012-01-01

    Aptamers generated from randomized libraries of nucleic acids have found utility in a wide variety of fields and in the clinic. Aptamers can be used to target both intracellular and extracellular components, including small molecules, proteins, cells, and viruses. With recent technological developments in stringent selection and rapid isolation strategies, it is likely that aptamers will continue to make an impact as useful tools and reagents. Although many recently developed aptamers are int...

  11. Development of an efficient targeted cell-SELEX procedure for DNA aptamer reagents.

    Directory of Open Access Journals (Sweden)

    Susanne Meyer

    Full Text Available BACKGROUND: DNA aptamers generated by cell-SELEX offer an attractive alternative to antibodies, but generating aptamers to specific, known membrane protein targets has proven challenging, and has severely limited the use of aptamers as affinity reagents for cell identification and purification. METHODOLOGY: We modified the BJAB lymphoblastoma cell line to over-express the murine c-kit cell surface receptor. After six rounds of cell-SELEX, high-throughput sequencing and bioinformatics analysis, we identified aptamers that bound BJAB cells expressing c-kit but not wild-type BJAB controls. One of these aptamers also recognizes c-kit endogenously expressed by a mast cell line or hematopoietic progenitor cells, and specifically blocks binding of the c-kit ligand stem cell factor (SCF. This aptamer enables better separation by fluorescence-activated cell sorting (FACS of c-kit(+ hematopoietic progenitor cells from mixed bone marrow populations than a commercially available antibody, suggesting that this approach may be broadly useful for rapid isolation of affinity reagents suitable for purification of other specific cell types. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel procedure for the efficient generation of DNA aptamers that bind to specific cell membrane proteins and can be used as high affinity reagents. We have named the procedure STACS (Specific TArget Cell-SELEX.

  12. Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

    Science.gov (United States)

    Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si; Wang, Jin; Huang, Nan

    2016-11-01

    Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

  13. Isolation and Characterization of 2'-amino-modified RNA Aptamers for Human TNFα

    Institute of Scientific and Technical Information of China (English)

    Xinrui Yan; Xuwen Gao; Zhiqing Zhang

    2004-01-01

    Human tumor necrosis factor α (hTNFα), a pleiotropic cytokine with activities ranging from host defense mechanisms in infection and injury to severe toxicity in septic shock or other related diseases, is a promising target for drug screening. Using the SELEX (systematic evolution of ligands by exponential enrichment) process, we isolated oligonucleotide ligands (aptamers) with high affinities for hTNFα. Aptamers were selected from a starting pool of 40 randomized sequences composed of about 1015 RNA molecules. Representative aptamers were truncated to the minimal length with high affinity for hTNFα and were further modified by replacement of 2'-OH with 2'-F and 2'-NH2 at all ribopurine positions. These modified RNA aptamers were resistant to nuclease. The specificity of these aptamers for hTNFαwas confirmed, and their activity to inhibit the cytotoxicity of hTNFα on mouse L929 cells was determined. Results demonstrated that four 2'-NH2-modified aptamers bound to hTNFα with high affinity and blocked the binding of hTNFα to its receptor, thus protecting the L929 cells from the cytotoxicity of hTNFα. Oligonucleotide aptamers described here are potential therapeutics and diagnostics for hTNFc-related diseases.

  14. Small organic molecules detection based on aptamer-modified gold nanoparticles-enhanced quartz crystal microbalance with dissipation biosensor.

    Science.gov (United States)

    Zheng, Bin; Cheng, Sheng; Liu, Wei; Lam, Michael Hon-Wah; Liang, Haojun

    2013-07-15

    Small molecules are difficult to detect by the conventional quartz crystal microbalance with dissipation (QCM-D) technique directly because the changes in frequency resulting from the binding processes of small biomolecules are often small. In the current study, an aptamer-based gold nanoparticles (AuNPs)-enhanced sensing strategy for detection of small molecules was developed. The QCM crystal was first modified with a layer of thiolated linker DNA, which can be partly base-paired with the detection part containing the adenosine aptamer sequence. In the presence of adenosine, the aptamer bound with adenosine and folded to the complex structure, which precluded the reporter part carrying AuNPs to combine with the random coiled detection part. Therefore, the lower the concentration of adenosine, the more AuNPs combined to the crystal. The resulting aptasensor showed a linear response to the increase of the adenosine concentration in the range of 0-2 μM with a linear correlation of r=0.99148 and a detection limit of 65 nM. Moreover, the aptasensor exhibited several excellent characteristics such as high sensitivity, selectivity, good stability, and reproducibility.

  15. DNA aptamers for the detection of Haemophilus influenzae type b by cell SELEX.

    Science.gov (United States)

    Bitaraf, F S; Rasooli, I; Mousavi Gargari, S L

    2016-03-01

    Haemophilus influenzae type b (Hib) causes acute bacterial meningitis (ABM) in children, with a mortality rate of about 3-6 % of the affected patients. ABM can lead to death during a period of hours to several days and, hence, rapid and early detection of the infection is crucial. Aptamers, the short single-stranded DNA or RNA with high affinity to target molecules, are selected by a high-flux screening technique known as in vitro screening and systematic evolution of ligands by exponential enrichment technology (SELEX). In this study, whole-cell SELEX was applied for the selection of target-specific aptamers with high affinity to Hib. ssDNA aptamers prepared by lambda exonuclease were incubated with the target cells (Hib). The aptameric binding rate to Hib was characterized for binding affinity after seven SELEX rounds by flow cytometry. The aptamers with higher binding affinity were cloned. Four of 68 aptamer clones were selected for sequencing. The dissociation constant (Kd) of the high-affinity aptamer clones 45 and 63 were 47.10 and 28.46 pM, respectively. These aptamers did not bind to other bacterial species, including the seven meningitis-causing bacteria. They showed distinct affinity to various H. influenzae strains only. These aptamers showed the highest affinity to Hib and the lowest affinity to H. influenzae type c and to other meningitis-causing bacteria. Clone 63 could detect Hib in patients' cerebrospinal fluid (CSF) samples at 60 colony-forming units (CFU)/mL. The results indicate applicability of the aptamers for rapid and early detection of infections brought about by Hib.

  16. Selection of an aptamer antidote to the anticoagulant drug bivalirudin.

    Science.gov (United States)

    Martin, Jennifer A; Parekh, Parag; Kim, Youngmi; Morey, Timothy E; Sefah, Kwame; Gravenstein, Nikolaus; Dennis, Donn M; Tan, Weihong

    2013-01-01

    Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT). Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug), and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction) and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 µM aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care.

  17. Selection of an aptamer antidote to the anticoagulant drug bivalirudin.

    Directory of Open Access Journals (Sweden)

    Jennifer A Martin

    Full Text Available Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT. Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug, and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 µM aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care.

  18. Developing aptamers into tumor diagnostics and therapeutics

    Institute of Scientific and Technical Information of China (English)

    Jing Mi; Bryan M. Clary; Bruce A. Sullenger

    2008-01-01

    Aptamers are small single-stranded nucleic acid molecules that bind a target protein with high affinity and specificity. Due to their stability, low toxicity and immunogenicity, as well as improved safety, aptamers are attractive alternatives to antibody and are therefore suitable for in vivo applications. Aptamers are typically isolated, through a process termed SELEX (systematic evolution of ligands by exponential enrichment), from combinatorial libraries with desired proteins. In the present review, the recent non-conventional aptamer selection process will be discussed together with an overview on the aptamer application in cancer diagnosis and therapy.

  19. Electrochemical biosensors and logic devices based on aptamers

    Institute of Scientific and Technical Information of China (English)

    Zuo Xiaolei; Lin Meihua; Fan Chunhai

    2013-01-01

    Aptamers are molecular recognition elements with high specificity that are selected from deoxyribonucleic acid/ribonucleic acid (DNA/RNA) library.Compared with the traditional protein recognition elements,aptamers have excellent properties such as cost-effective,stable,easy for synthesis and modification.In recent years,electrochemistry plays an important role in biosensor field because of its high sensitivity,high stability,fast response and easy miniaturization.Through the combination of these two technologies and our rational design,we constructed a series of biosensors and biochips that are simple,fast,cheap and miniaturized.Firstly,we designed an adenosine triphosphate (ATP) electrochemical biosensor based on the strand displacement strategy.We can detect as low as 10 nmol/L of ATP both in pure solution and complicated cell lysates.Secondly,we creatively split the aptamers into two fragments and constructed the sandwich assay platform only based on single aptamer sequence.We successfully transferred this design on biochips with multiple micro electrodes (6×6) and accomplished multiplex detection.In the fields of biochips and biocomputers,we designed several DNA logic gates with electric (electrochemical) signal as output which paves a new way for the development of DNA computer.

  20. Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers

    Science.gov (United States)

    Pica, Andrea; Russo Krauss, Irene; Parente, Valeria; Tateishi-Karimata, Hisae; Nagatoishi, Satoru; Tsumoto, Kouhei; Sugimoto, Naoki; Sica, Filomena

    2017-01-01

    Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed. PMID:27899589

  1. Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers.

    Science.gov (United States)

    Pica, Andrea; Russo Krauss, Irene; Parente, Valeria; Tateishi-Karimata, Hisae; Nagatoishi, Satoru; Tsumoto, Kouhei; Sugimoto, Naoki; Sica, Filomena

    2017-01-09

    Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed.

  2. In vitro selection of G-rich RNA aptamers that target HIV-1 integrase

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Aptamers that interact with various HIV-1 proteins,such as reverse transcriptase,Rev,Tat protein,and nuclear capsule protein,have been prepared through SELEX (systematic evolution of ligands by ex-ponential enrichment) technique. However,there are few reports about the DNA or RNA aptamers that target HIV-1 integrase. In this investigation,we selected alternative RNA aptamers specific for the HIV-1 integrase by using a different binding buffer containing 10 mmol·L-1 MgCl2 and 100 mmol·L-1 KCl. Aptamer IN1,IN2,IN3 had similar and the highest Kd values from 145 to 239 nmol·L-1. Structural studies showed that they formed similar stem-loop structure. Deletion of any stem structure resulted in diminished affinity. In addition,structure probing study with antisense DNA indicated that the stem-loop structure in the random region was critical for integrase binding. Although aptamer IN1 failed to form G-quartet structure,it might directly interact with the DDE motif of integrase,which is the virus DNA-binding site,because G-quadruplex T40214 competitively inhibited the interaction between IN1 and integrase. Together,this study generated a novel RNA aptamer IN1,which could be useful in basic research and anti-HIV drug screening.

  3. In vitro selection of G-rich RNA aptamers that target HIV-1 integrase

    Institute of Scientific and Technical Information of China (English)

    LIU YingChun; ZHANG Yan; YE GuoZhu; YANG ZhenJun; ZHANG LiangRen; ZHANG LiHe

    2008-01-01

    Aptamers that interact with various HIV-1 proteins, such as reverse transcriptase, Rev, Tat protein, and nuclear capsule protein, have been prepared through SELEX (systematic evolution of ligands by ex-ponential enrichment) technique. However, there are few reports about the DNA or RNA aptamers that target HIV-1 integrase. In this investigation, we selected alternative RNA aptamers specific for the HIV-1 Aptamer IN1, IN2, IN3 had similar and the highest Kd values from 145 to 239 nmol. L-1. Structural studies showed that they formed similar stem-loop structure. Deletion of any stem structure resulted in diminished affinity. In addition, structure probing study with antisense DNA indicated that the stem-loop structure in the random region was critical for integrase binding. Although aptamer IN1 failed to form G-quartet structure, it might directly interact with the DDE motif of integrase, which is the virus DNA-binding site, because G-quadruplex T40214 competitively inhibited the interaction between IN1 and integrase. Together, this study generated a novel RNA aptamer IN1, which could be useful in basic research and anti-HIV drug screening.

  4. Tools for integrated sequence-structure analysis with UCSF Chimera

    Directory of Open Access Journals (Sweden)

    Huang Conrad C

    2006-07-01

    Full Text Available Abstract Background Comparing related structures and viewing the structures in the context of sequence alignments are important tasks in protein structure-function research. While many programs exist for individual aspects of such work, there is a need for interactive visualization tools that: (a provide a deep integration of sequence and structure, far beyond mapping where a sequence region falls in the structure and vice versa; (b facilitate changing data of one type based on the other (for example, using only sequence-conserved residues to match structures, or adjusting a sequence alignment based on spatial fit; (c can be used with a researcher's own data, including arbitrary sequence alignments and annotations, closely or distantly related sets of proteins, etc.; and (d interoperate with each other and with a full complement of molecular graphics features. We describe enhancements to UCSF Chimera to achieve these goals. Results The molecular graphics program UCSF Chimera includes a suite of tools for interactive analyses of sequences and structures. Structures automatically associate with sequences in imported alignments, allowing many kinds of crosstalk. A novel method is provided to superimpose structures in the absence of a pre-existing sequence alignment. The method uses both sequence and secondary structure, and can match even structures with very low sequence identity. Another tool constructs structure-based sequence alignments from superpositions of two or more proteins. Chimera is designed to be extensible, and mechanisms for incorporating user-specific data without Chimera code development are also provided. Conclusion The tools described here apply to many problems involving comparison and analysis of protein structures and their sequences. Chimera includes complete documentation and is intended for use by a wide range of scientists, not just those in the computational disciplines. UCSF Chimera is free for non-commercial use and is

  5. Monitoring genomic sequences during SELEX using high-throughput sequencing: neutral SELEX.

    Directory of Open Access Journals (Sweden)

    Bob Zimmermann

    Full Text Available BACKGROUND: SELEX is a well established in vitro selection tool to analyze the structure of ligand-binding nucleic acid sequences called aptamers. Genomic SELEX transforms SELEX into a tool to discover novel, genomically encoded RNA or DNA sequences binding a ligand of interest, called genomic aptamers. Concerns have been raised regarding requirements imposed on RNA sequences undergoing SELEX selection. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate SELEX and assess the extent of these effects, we designed and performed a Neutral SELEX experiment omitting the selection step, such that the sequences are under the sole selective pressure of SELEX's amplification steps. Using high-throughput sequencing, we obtained thousands of full-length sequences from the initial genomic library and the pools after each of the 10 rounds of Neutral SELEX. We compared these to sequences obtained from a Genomic SELEX experiment deriving from the same initial library, but screening for RNAs binding with high affinity to the E. coli regulator protein Hfq. With each round of Neutral SELEX, sequences became less stable and changed in nucleotide content, but no sequences were enriched. In contrast, we detected substantial enrichment in the Hfq-selected set with enriched sequences having structural stability similar to the neutral sequences but with significantly different nucleotide selection. CONCLUSIONS/SIGNIFICANCE: Our data indicate that positive selection in SELEX acts independently of the neutral selective requirements imposed on the sequences. We conclude that Genomic SELEX, when combined with high-throughput sequencing of positively and neutrally selected pools, as well as the gnomic library, is a powerful method to identify genomic aptamers.

  6. Aptamer-based multiplexed proteomic technology for biomarker discovery.

    Directory of Open Access Journals (Sweden)

    Larry Gold

    Full Text Available BACKGROUND: The interrogation of proteomes ("proteomics" in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. METHODOLOGY/PRINCIPAL FINDINGS: We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma. Our current assay measures 813 proteins with low limits of detection (1 pM median, 7 logs of overall dynamic range (~100 fM-1 µM, and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD. We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. CONCLUSIONS/SIGNIFICANCE: We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next

  7. Nonlinear Correlations of Protein Sequences and Symmetries of Their Structures

    Institute of Scientific and Technical Information of China (English)

    LI Ming-Feng; HUANG Yan-Zhao; XIAO Yi

    2005-01-01

    @@ We investigate the nonlinear correlations of protein sequences by using the nonlinear prediction method developed in nonlinear dynamical theory.It is found that a lot of protein sequences show strong nonlinear correlations and have deterministic structures.Further investigations show that the strong nonlinear correlations of these protein sequences are due to the symmetries of their tertiary structures.Furthermore, the correlation lengths of the sequences are related to the degrees of the symmetries.These results support the duplication mechanism of protein evolution and also reveal one aspect how amino acid sequences encode their spatial structures.

  8. Finding Common Sequence and Structure Motifs in a set of RNA sequences

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Heyer, Laurie J.; Stormo, Gary D.

    1997-01-01

    We present a computational scheme to search for the most common motif, composed of a combination of sequence and structure constraints, among a collection of RNA sequences. The method uses a simplified version of the Sankoff algorithm for simultaneous folding and alignment of RNA sequences...

  9. Identification of protein superfamily from structure- based sequence motif

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The structure-based sequence motif of the distant proteins in evolution, protein tyrosine phosphatases (PTP) Ⅰ and Ⅱ superfamilies, as an example, has been defined by the structural comparison, structure-based sequence alignment and analyses on substitution patterns of residues in common sequence conserved regions. And the phosphatases Ⅰ and Ⅱ can be correctly identified together by the structure-based PTP sequence motif from SWISS-PROT and TrEBML databases. The results show that the correct rates of identification are over 98%. This is the first time to identify PTP Ⅰ and Ⅱ together by this motif.

  10. Quantum dot-DNA aptamer conjugates coupled with capillary electrophoresis: A universal strategy for ratiometric detection of organophosphorus pesticides.

    Science.gov (United States)

    Tang, Tingting; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

    2016-01-01

    Based on the highly sensitivity and stable-fluorescence of water-soluble CdTe/CdS core-shell quantum dots (QDs) with broad-specificity DNA aptamers, a novel ratiometric detection strategy was proposed for the sensitive detection of organophosphorus pesticides by capillary electrophoresis with laser-induced fluorescence (CE-LIF). The as-prepared QDs were first conjugated with the amino-modified oligonucleotide (AMO) by amidation reaction, which is partial complementary to the DNA aptamer of organophosphorus pesticides. Then QD-labeled AMO (QD-AMO) was incubated with the DNA aptamer to form QD-AMO-aptamer duplex. When the target organophosphorus pesticides were added, they could specifically bind the DNA aptamer, leading to the cleavage of QD-AMO-aptamer duplex, accompany with the release of QD-AMO. As a result, the ratio of peak height between QD-AMO and QD-AMO-aptamer duplex changed in the detection process of CE-LIF. This strategy was subsequently applied for the detection of phorate, profenofos, isocarbophos, and omethoate with the detection limits of 0.20, 0.10, 0.17, and 0.23μM, respectively. This is the first report about using QDs as the signal indicators for organophosphorus pesticides detection based on broad-specificity DNA aptamers by CE-LIF, thus contributing to extend the scope of application of QDs in different fields. The proposed method has great potential to be a universal strategy for rapid detection of aptamer-specific small molecule targets by simply changing the types of aptamer sequences.

  11. Theophylline detection using an aptamer and DNA-gold nanoparticle conjugates.

    Science.gov (United States)

    Chávez, Jorge L; Lyon, Wanda; Kelley-Loughnane, Nancy; Stone, Morley O

    2010-09-15

    A detection system for theophylline that combined the recognition properties of an aptamer and the plasmonic response of gold nanoparticles (AuNPs) is presented. The aptamer was used as a linker for AuNPs functionalized with complementary sequences to the aptamer (DNA-AuNPs), producing supramolecular complexes that disassemble when exposed to theophylline due to aptamer binding. The detection event was reported as a change in the AuNPs plasmonic peak and intensity. Addition of a spacer on the DNA immobilized on the AuNPs facing the aptamer binding site improved the aggregates' response, doubling the detection range of system response to theophylline. Modification of the oligonucleotides immobilized on the AuNPs that reduced the interparticle distance in the aggregated state suppressed their response to theophylline and addition of the spacer recovered it. This work demonstrated that the design of oligonucleotides immobilized on the AuNPs could be used to improve their plasmonic response without affecting aptamer performance.

  12. Morph-X-Select: Morphology-based tissue aptamer selection for ovarian cancer biomarker discovery

    Science.gov (United States)

    Wang, Hongyu; Li, Xin; Volk, David E.; Lokesh, Ganesh L.-R.; Elizondo-Riojas, Miguel-Angel; Li, Li; Nick, Alpa M.; Sood, Anil K.; Rosenblatt, Kevin P.; Gorenstein, David G.

    2016-01-01

    High affinity aptamer-based biomarker discovery has the advantage of simultaneously discovering an aptamer affinity reagent and its target biomarker protein. Here, we demonstrate a morphology-based tissue aptamer selection method that enables us to use tissue sections from individual patients and identify high-affinity aptamers and their associated target proteins in a systematic and accurate way. We created a combinatorial DNA aptamer library that has been modified with thiophosphate substitutions of the phosphate ester backbone at selected 5′dA positions for enhanced nuclease resistance and targeting. Based on morphological assessment, we used image-directed laser microdissection (LMD) to dissect regions of interest bound with the thioaptamer (TA) library and further identified target proteins for the selected TAs. We have successfully identified and characterized the lead candidate TA, V5, as a vimentin-specific sequence that has shown specific binding to tumor vasculature of human ovarian tissue and human microvascular endothelial cells. This new Morph-X-Select method allows us to select high-affinity aptamers and their associated target proteins in a specific and accurate way, and could be used for personalized biomarker discovery to improve medical decision-making and to facilitate the development of targeted therapies to achieve more favorable outcomes. PMID:27839510

  13. Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Wang, Jin, E-mail: jinxxwang@263.net [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); Huang, Nan [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)

    2016-11-15

    Highlights: • The dopamine/PEI film with controlled amine density was successfully prepared. • The DNA aptamer was assembled onto the film via electrostatic incorporation. • The A@DPfilmscanspecificallyandeffectivelycaptureEPCs. • The A@DP film can support the survival of ECs, control the hyperplasia of SMCs. • The dynamic/co-culture models are useful for studying cells competitive adhesion. - Abstract: Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

  14. Aptamer-based label-free impedimetric biosensor for detection of progesterone.

    Science.gov (United States)

    Contreras Jiménez, Gastón; Eissa, Shimaa; Ng, Andy; Alhadrami, Hani; Zourob, Mohammed; Siaj, Mohamed

    2015-01-20

    Rising progesterone (P4) levels in humans due to its overconsumption through hormonal therapy, food products, or drinking water can lead to many negative health effects. Thus, the simple and accurate assessment of P4 in both environmental and clinical samples is highly important to protect public health. In this work, we present the selection, identification, and characterization of ssDNA aptamers with high binding affinity to P4. The aptamers were selected in vitro from a single-stranded DNA library of 1.8 × 10(15) oligonucleotides showing dissociation constants (KD) in the low nanomolar range. The dissociation constant of the best aptamer, designated as P4G13, was estimated to be 17 nM by electrochemical impedance spectroscopy (EIS) as well as fluorometric assay. Moreover, the aptamer P4G13 did not show cross-reactivity to analogues similar to progesterone such as 17β-estradiol (E2) and norethisterone (NET). An impedimetric aptasensor for progesterone was then fabricated based on the conformational change of P4G13 aptamer, immobilized on the gold electrode by self-assembly, upon binding to P4, which results in an increase in electron transfer resistance. Aptamer-complementary DNA (cDNA) oligonucleotides were tested to maximize the signal gain of the aptasensor after binding with progesterone. Significant signal enhancement was observed when the aptamer hybridized with a short complementary sequence at specific site was used instead of pure aptamer. This signal gain is likely due to the more significant conformational change of the aptamer-cDNA than the pure aptamer upon binding with P4, as confirmed by circular dichroism (CD) spectroscopy. The developed aptasensor exhibited a linear range for concentrations of P4 from 10 to 60 ng/mL with a detection limit of 0.90 ng/mL. Moreover, the aptasensor was applied in spiked tap water samples and showed good recovery percentages. The new selected progesterone aptamers can be exploited in further biosensing applications

  15. Label-free aptamer biosensor for selective detection of thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Na, Weidan; Liu, Xiaotong; Wang, Lei; Su, Xingguang, E-mail: suxg@jlu.edu.cn

    2015-10-29

    We fabricated a novel fluorescence biosensor for the selective detection of thrombin by using bovine serum albumin-capped CdS quantum dots (BSA-CdS QDs). Two kinds of designed DNA (DNA1 and DNA2) could bind to CdS QDs through the electrostatic interaction between DNA and Cd{sup 2+} on the surface of CdS QDs. The obtained DNA/BSA-CdS QDs kept stable in the solution with the fluorescence intensity obviously enhanced. Hairpin structure of DNA1contained two domains, one is the aptamer sequence of thrombin and the other is the complementary sequence of DNA2. When thrombin was added, it would bind to DNA1 and induce the hairpin structure of DNA1 changed into G-quadplex structure. Meanwhile, DNA2 would transfer from the surface of CdS QDs to DNA1 via hybridization, which resulted in the removal of DNA1 and DNA2 from the surface of CdS QDs, and led to the fluorescence intensity of CdS QDs reduced. Thus, the determination of thrombin could be achieved by monitoring the change of the fluorescence intensity of CdS QDs. The present method is simple and fast, and exhibits good selectivity for thrombin over other proteins. We have successfully detected thrombin in human serum samples with satisfactory results. - Highlights: • A novel strategy for the detection of thrombin was established based on BSA-CdS QDs. • DNA could serve as the co-ligands to stabilize CdS QDs and enhance the fluorescence intensity. • Thrombin could change the structure of DNA1 and quench the fluorescence of CdS QDs. • Thrombin in real sample was detected with satisfactory results.

  16. PSSARD: protein sequence-structure analysis relational database.

    Science.gov (United States)

    Guruprasad, Kunchur; Srikanth, K; Babu, A V N

    2005-09-15

    We have implemented a relational database comprising a representative dataset of amino acid sequences and their associated secondary structure. The representative amino acid sequences were selected according to the PDB_SELECT program by choosing proteins corresponding to protein crystal structure data deposited in the protein data bank that share less than 25% overall pair-wise sequence identity. The secondary structure was extracted from the protein data bank website. The information content in the database includes the protein description, PDB code, crystal structure resolution, total number of amino acid residues in the protein chain, amino acid sequence, secondary structure conformation and its summary. The database is freely accessible from the website mentioned below and is useful to query on any of the above fields. The database is particularly useful to quickly retrieve amino acid sequences that are compatible to any super-secondary structure conformation from several proteins simultaneously.

  17. Dynamics in Sequence Space for RNA Secondary Structure Design.

    Science.gov (United States)

    Matthies, Marco C; Bienert, Stefan; Torda, Andrew E

    2012-10-01

    We have implemented a method for the design of RNA sequences that should fold to arbitrary secondary structures. A popular energy model allows one to take the derivative with respect to composition, which can then be interpreted as a force and used for Newtonian dynamics in sequence space. Combined with a negative design term, one can rapidly sample sequences which are compatible with a desired secondary structure via simulated annealing. Results for 360 structures were compared with those from another nucleic acid design program using measures such as the probability of the target structure and an ensemble-weighted distance to the target structure.

  18. Aptamers overview: selection, features and applications.

    Science.gov (United States)

    Hernandez, Luiza I; Machado, Isabel; Schafer, Thomas; Hernandez, Frank J

    2015-01-01

    Apatamer technology has been around for a quarter of a century and the field had matured enough to start seeing real applications, especially in the medical field. Since their discovery, aptamers rapidly emerged as key players in many fields, such as diagnostics, drug discovery, food science, drug delivery and therapeutics. Because of their synthetic nature, aptamers are evolving at an exponential rate gaining from the newest advances in chemistry, nanotechnology, biology and medicine. This review is meant to give an overview of the aptamer field, by including general aspects of aptamer identification and applications as well as highlighting certain features that contribute to their quick deployment in the biomedical field.

  19. Formatt: Correcting protein multiple structural alignments by incorporating sequence alignment

    Directory of Open Access Journals (Sweden)

    Daniels Noah M

    2012-10-01

    Full Text Available Abstract Background The quality of multiple protein structure alignments are usually computed and assessed based on geometric functions of the coordinates of the backbone atoms from the protein chains. These purely geometric methods do not utilize directly protein sequence similarity, and in fact, determining the proper way to incorporate sequence similarity measures into the construction and assessment of protein multiple structure alignments has proved surprisingly difficult. Results We present Formatt, a multiple structure alignment based on the Matt purely geometric multiple structure alignment program, that also takes into account sequence similarity when constructing alignments. We show that Formatt outperforms Matt and other popular structure alignment programs on the popular HOMSTRAD benchmark. For the SABMark twilight zone benchmark set that captures more remote homology, Formatt and Matt outperform other programs; depending on choice of embedded sequence aligner, Formatt produces either better sequence and structural alignments with a smaller core size than Matt, or similarly sized alignments with better sequence similarity, for a small cost in average RMSD. Conclusions Considering sequence information as well as purely geometric information seems to improve quality of multiple structure alignments, though defining what constitutes the best alignment when sequence and structural measures would suggest different alignments remains a difficult open question.

  20. Protein folds and families: sequence and structure alignments.

    Science.gov (United States)

    Holm, L; Sander, C

    1999-01-01

    Dali and HSSP are derived databases organizing protein space in the structurally known regions. We use an automatic structure alignment program (Dali) for the classification of all known 3D structures based on all-against-all comparison of 3D structures in the Protein Data Bank. The HSSP database associates 1D sequences with known 3D structures using a position-weighted dynamic programming method for sequence profile alignment (MaxHom). As a result, the HSSP database not only provides aligned sequence families, but also implies secondary and tertiary structures covering 36% of all sequences in Swiss-Prot. The structure classification by Dali and the sequence families in HSSP can be browsed jointly from a web interface providing a rich network of links between neighbours in fold space, between domains and proteins, and between structures and sequences. In particular, this results in a database of explicit multiple alignments of protein families in the twilight zone of sequence similarity. The organization of protein structures and families provides a map of the currently known regions of the protein universe that is useful for the analysis of folding principles, for the evolutionary unification of protein families and for maximizing the information return from experimental structure determination. The databases are available from http://www.embl-ebi.ac.uk/dali/

  1. Selection of aptamers for use as radiopharmaceuticals in bacterial infection diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Ieda Mendes; Faria, Ligia Santana de; Correa, Cristiane Rodrigues; Andrade, Antero Silva Ribeiro de, E-mail: imendesf@yahoo.com.br, E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)

    2013-07-01

    The difficulty in early detection of specific foci in the bacterial infection caused by bacteria has raised the need to search for new techniques for this purpose, since these foci require prolonged treatment with antibiotics and in some cases even drainage or, if applicable, removal of prostheses or grafts. Detection of bacterial infections by scintigraphy has the advantage that an image of the whole body could be obtained. This study aims to obtain aptamers specific bacteria for future use as radiopharmaceutical. The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) methodology can generate oligonucleotides (aptamers) that are able to bind with high affinity and specificity to a specific target, from small molecules to complex proteins, by using rounds of enrichment and amplification. Aptamers can be labeled with different radionucleotides such as {sup 99m}Tc, {sup 18}F and {sup 32}P. In this study aptamers anti-peptidoglycan, the main component of the outer cell wall of bacteria, were obtained through SELEX. The SELEX started with a pool of ssDNA that had 10{sup 15}different sequences (library), each oligo has two fixed regions merging a portion of 25 random nucleotides. Initially, the library of ssDNA was incubated with peptidoglycan, for 1h at 37 dec C with stirring. Subsequently, amplification of oligonucleotides that were able to bind to peptidoglycan was performed by PCR (Polymerase Chain Reaction). The amplified oligonucleotides were again incubated with peptidoglycan, amplified and purified. At the end of 15 rounds of selection the oligonucleotides were cloned using TOPO plasmid and Escherichia coli strain Top10F'. The plasmid DNA from 40 colonies were extracted and quantified. The plasmids were sequenced using the sequencing MegaBase, and two different aptamers sequences were obtained from all clones. The aptamers obtained were synthesized and subsequently labeled with {sup 32}P in the 5' end. The labeled aptamers were incubated

  2. Use of cell-SELEX to generate DNA aptamers as molecular probes of HPV-associated cervical cancer cells.

    Directory of Open Access Journals (Sweden)

    Jessica C Graham

    Full Text Available BACKGROUND: Disease-specific biomarkers are an important tool for the timely and effective management of pathological conditions, including determination of susceptibility, diagnosis, and monitoring efficacy of preventive or therapeutic strategies. Aptamers, comprising single-stranded or double-stranded DNA or RNA, can serve as biomarkers of disease or biological states. Aptamers can bind to specific epitopes on macromolecules by virtue of their three dimensional structures and, much like antibodies, aptamers can be used to target specific epitopes on the basis of their molecular shape. The Systematic Evolution of Ligands by EXponential enrichment (SELEX is the approach used to select high affinity aptamers for specific macromolecular targets from among the >10(13 oligomers comprising typical random oligomer libraries. In the present study, we used live cell-based SELEX to identify DNA aptamers which recognize cell surface differences between HPV-transformed cervical carcinoma cancer cells and isogenic, nontumorigenic, revertant cell lines. METHODOLOGY/PRINCIPAL FINDINGS: Whole-cell SELEX methodology was adapted for use with adherent cell lines (which we termed Adherent Cell-SELEX (AC-SELEX. Using this approach, we identified high affinity aptamers (nanomolar range K(d to epitopes specific to the cell surface of two nontumorigenic, nontumorigenic revertants derived from the human cervical cancer HeLa cell line, and demonstrated the loss of these epitopes in another human papillomavirus transformed cervical cancer cell line (SiHa. We also performed preliminary investigation of the aptamer epitopes and their binding characteristics. CONCLUSIONS/SIGNIFICANCE: Using AC-SELEX we have generated several aptamers that have high affinity and specificity to the nontumorigenic, revertant of HPV-transformed cervical cancer cells. These aptamers can be used to identify new biomarkers that are related to carcinogenesis. Panels of aptamers, such as these may

  3. Evolutionary optimization of biopolymers and sequence structure maps

    Energy Technology Data Exchange (ETDEWEB)

    Reidys, C.M.; Kopp, S.; Schuster, P. [Institut fuer Molekulare Biotechnologie, Jena (Germany)

    1996-06-01

    Searching for biopolymers having a predefined function is a core problem of biotechnology, biochemistry and pharmacy. On the level of RNA sequences and their corresponding secondary structures we show that this problem can be analyzed mathematically. The strategy will be to study the properties of the RNA sequence to secondary structure mapping that is essential for the understanding of the search process. We show that to each secondary structure s there exists a neutral network consisting of all sequences folding into s. This network can be modeled as a random graph and has the following generic properties: it is dense and has a giant component within the graph of compatible sequences. The neutral network percolates sequence space and any two neutral nets come close in terms of Hamming distance. We investigate the distribution of the orders of neutral nets and show that above a certain threshold the topology of neutral nets allows to find practically all frequent secondary structures.

  4. Nucleosome DNA sequence structure of isochores

    Directory of Open Access Journals (Sweden)

    Trifonov Edward N

    2011-04-01

    Full Text Available Abstract Background Significant differences in G+C content between different isochore types suggest that the nucleosome positioning patterns in DNA of the isochores should be different as well. Results Extraction of the patterns from the isochore DNA sequences by Shannon N-gram extension reveals that while the general motif YRRRRRYYYYYR is characteristic for all isochore types, the dominant positioning patterns of the isochores vary between TAAAAATTTTTA and CGGGGGCCCCCG due to the large differences in G+C composition. This is observed in human, mouse and chicken isochores, demonstrating that the variations of the positioning patterns are largely G+C dependent rather than species-specific. The species-specificity of nucleosome positioning patterns is revealed by dinucleotide periodicity analyses in isochore sequences. While human sequences are showing CG periodicity, chicken isochores display AG (CT periodicity. Mouse isochores show very weak CG periodicity only. Conclusions Nucleosome positioning pattern as revealed by Shannon N-gram extension is strongly dependent on G+C content and different in different isochores. Species-specificity of the pattern is subtle. It is reflected in the choice of preferentially periodical dinucleotides.

  5. Insights into ligand binding to PreQ1 Riboswitch Aptamer from molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Zhou Gong

    Full Text Available Riboswitches play roles in transcriptional or translational regulation through specific ligand binding of their aptamer domains. Although a number of ligand-bound aptamer complex structures have been solved, it is important to know ligand-free conformations of the aptamers in order to understand the mechanism of specific binding by ligands. In this paper, preQ1 riboswitch aptamer domain from Bacillus subtilis is studied by overall 1.5 μs all-atom molecular dynamics simulations We found that the ligand-free aptamer has a stable state with a folded P1-L3 and open binding pocket. The latter forms a cytosine-rich pool in which the nucleotide C19 oscillates between close and open positions, making it a potential conformation for preQ1 entrance. The dynamic picture further suggests that the specific recognition of preQ1 by the aptamer domain is not only facilitated by the key nucleotide C19 but also aided and enhanced by other cytosines around the binding pocket. These results should help to understand the details of preQ1 binding.

  6. Aptamers to the sigma factor mimic promoter recognition and inhibit transcription initiation by bacterial RNA polymerase.

    Science.gov (United States)

    Miropolskaya, Nataliya; Kulbachinskiy, Andrey

    2016-01-08

    Promoter recognition by bacterial RNA polymerase (RNAP) is a multi-step process involving multiple protein-DNA interactions and several structural and kinetic intermediates which remain only partially characterized. We used single-stranded DNA aptamers containing specific promoter motifs to probe the interactions of the Thermus aquaticus RNAP σ(A) subunit with the -10 promoter element in the absence of other parts of the promoter complex. The aptamer binding decreased intrinsic fluorescence of the σ subunit, likely as a result of interactions between the -10 element and conserved tryptophan residues of the σ DNA-binding region 2. By monitoring these changes, we demonstrated that DNA binding proceeds through a single rate-limiting step resulting in formation of very stable complexes. Deletion of the N-terminal domain of the σ(A) subunit increased the rate of aptamer binding while replacement of this domain with an unrelated N-terminal region 1.1 from the Escherichia coli σ(70) subunit restored the original kinetics of σ-aptamer interactions. The results demonstrate that the key step in promoter recognition can be modelled in a simple σ-aptamer system and reveal that highly divergent N-terminal domains similarly modulate the DNA-binding properties of the σ subunit. The aptamers efficiently suppressed promoter-dependent transcription initiation by the holoenzyme of RNA polymerase, suggesting that they may be used for development of novel transcription inhibitors.

  7. Generation and characterization of quinolone-specific DNA aptamers suitable for water monitoring.

    Science.gov (United States)

    Reinemann, C; Freiin von Fritsch, U; Rudolph, S; Strehlitz, B

    2016-03-15

    Quinolones are antibiotics that are accredited in human and veterinary medicine but are regularly used in high quantities also in industrial livestock farming. Since these compounds are often only incompletely metabolized, significant amounts contaminate the aquatic environment and negatively impact on a variety of different ecosystems. Although there is increasing awareness of problems caused by pharmaceutical pollution, available methods for the detection and elimination of numerous pharmaceutical residues are currently inefficient or expensive. While this also applies to antibiotics that may lead to multi-drug resistance in pathogenic bacteria, aptamer-based technologies potentially offer alternative approaches for sensitive and efficient monitoring of pharmaceutical micropollutants. Using the Capture-SELEX procedure, we here describe the selection of an aptamer pool with enhanced binding qualities for fluoroquinolones, a widely used group of antibiotics in both human and veterinary medicine. The selected aptamers were shown to detect various quinolones with high specificity, while specific binding activities to structurally unrelated drugs were not detectable. The quinolone-specific aptamers bound to ofloxacin, one of the most frequently prescribed fluoroquinolone, with high affinity (KD=0.1-56.9 nM). The functionality of quinolone-specific aptamers in real water samples was demonstrated in local tap water and in effluents of sewage plants. Together, our data suggest that these aptamers may be applicable as molecular receptors in biosensors or as catcher molecules in filter systems for improved monitoring and treatment of polluted water.

  8. Plasmonic aptamer-gold nanoparticle sensors for small molecule fingerprint identification.

    Science.gov (United States)

    Chávez, Jorge L; Leny, Juliann K; Witt, Suzanne; Slusher, Grant M; Hagen, Joshua A; Kelley-Loughnane, Nancy

    2014-12-07

    The utilization of the plasmonic response of aptamer-gold nanoparticle conjugates (Apt-AuNPs) to design cross-reactive arrays for fingerprint identification of small molecular targets was demonstrated for the first time. Four aptamers with different structural features previously selected to bind different targets were used in combination with AuNPs by adsorbing the DNA on the AuNPs surface. The optimized response of the Apt-AuNPs to the analytes showed that, depending on the specific aptamer used, target binding by the aptamer could result in an increase or decrease of Apt-AuNPs stability. These Apt-AuNPs showed the ability to recognize different analytes with different affinities, generating fingerprints that allowed unambiguous analyte identification with response times in less than fifteen minutes. Importantly, it was observed that it was not necessary to select an aptamer per analyte of interest to generate differentiable signatures, but a subset of aptamers could be used to identify a larger number of analytes. The data was analyzed using principal component analysis, showing efficient clustering of the different datasets for qualitative and quantitative identification. This work opens the door to using these Apt-AuNPs in point of care diagnostics applications where fast sensors with easy to read outputs are needed.

  9. Finding the most significant common sequence and structure motifs in a set of RNA sequences

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Heyer, L.J.; Stormo, G.D.

    1997-01-01

    We present a computational scheme to locally align a collection of RNA sequences using sequence and structure constraints, In addition, the method searches for the resulting alignments with the most significant common motifs, among all possible collections, The first part utilizes a simplified ve...

  10. DNA-Aptamers Binding Aminoglycoside Antibiotics

    Directory of Open Access Journals (Sweden)

    Nadia Nikolaus

    2014-02-01

    Full Text Available Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

  11. Selection and characterization of DNA aptamers

    NARCIS (Netherlands)

    Ruigrok, V.J.B.

    2013-01-01

    This thesis focusses on the selection and characterisation of DNA aptamers and the various aspects related to their selection from large pools of randomized oligonucleotides. Aptamers are affinity tools that can specifically recognize and bind predefined target molecules; this ability, however, is n

  12. Using Aptamers for Cancer Biomarker Discovery

    Directory of Open Access Journals (Sweden)

    Yun Min Chang

    2013-01-01

    Full Text Available Aptamers are single-stranded synthetic DNA- or RNA-based oligonucleotides that fold into various shapes to bind to a specific target, which includes proteins, metals, and molecules. Aptamers have high affinity and high specificity that are comparable to that of antibodies. They are obtained using iterative method, called (Systematic Evolution of Ligands by Exponential Enrichment SELEX and cell-based SELEX (cell-SELEX. Aptamers can be paired with recent advances in nanotechnology, microarray, microfluidics, and other technologies for applications in clinical medicine. One particular area that aptamers can shed a light on is biomarker discovery. Biomarkers are important in diagnosis and treatment of cancer. In this paper, we will describe ways in which aptamers can be used to discover biomarkers for cancer diagnosis and therapeutics.

  13. 适配子识别技术在真菌毒素快速分析中的应用%Application of Aptamer Identification Technology in Rapid Analysis of Mycotoxins

    Institute of Scientific and Technical Information of China (English)

    杨锡辉; 孔维军; 杨美华; 赵明; 欧阳臻

    2013-01-01

    适配子是近几年来通过指数富集配基的系统进化技术(SELEX)体外筛选得到的一类与目标分子高特异性结合的寡聚核苷酸片段.适配子作为一个优于抗体的新型识别分子,已在食品快速检验、环境检测、新药研发、临床诊断和治疗以及毒理研究等领域展示了良好的应用前景.本文阐述了适配子识别靶物质的结构基础及其特性,归纳了目前已筛选出的真菌毒素适配子序列,总结了适配子在真菌毒素前处理,适配子光学分析技术和电化学分析技术在单种和多种真菌毒素检测中的应用突破,并展望了适配子识别技术在真菌毒素分析领域的发展趋势.%Aptamers are oligonucleotides fragments, such as ribonucleic acid ( RNA) and single-strand deoxyribonucleicacid ( ssDNA) or peptide molecules that can bind to their targets with high affinity and speci?ficity. For generation of artificial ligands, they are isolated from combinatorial libraries of synthetic nucleic acid by exponential enrichment, via an in vitro iterative process of adsorption, recovery and reamplification known as systematic evolution of ligands by exponential enrichment (SELEX). As new recognition molecules, aptamers display better prospects than antibodies in the fields of rapid analysis of food analysis, environmental monitoring, new drug development, clinical diagnosis and treatment, toxicological studies, etc. The structural bases, characteristics of combination between aptamers and analytes are elaborated in this article. Also, aptamer sequences of mytoxins are listed. Furhter, the application of aptamers identification technology for the rapid analysis of mycotoxins is summarized. These analytical applications involve extraction and purification of the sample, aptamer based optical analysis techniques and electrochemical analysis techniques for the rapid detection of single mycotoxin and simultaneous detection of various mycotoxins. Finally, the future

  14. Genomic SELEX: a discovery tool for genomic aptamers.

    Science.gov (United States)

    Zimmermann, Bob; Bilusic, Ivana; Lorenz, Christina; Schroeder, Renée

    2010-10-01

    Genomic SELEX is a discovery tool for genomic aptamers, which are genomically encoded functional domains in nucleic acid molecules that recognize and bind specific ligands. When combined with genomic libraries and using RNA-binding proteins as baits, Genomic SELEX used with high-throughput sequencing enables the discovery of genomic RNA aptamers and the identification of RNA-protein interaction networks. Here we describe how to construct and analyze genomic libraries, how to choose baits for selections, how to perform the selection procedure and finally how to analyze the enriched sequences derived from deep sequencing. As a control procedure, we recommend performing a "Neutral" SELEX experiment in parallel to the selection, omitting the selection step. This control experiment provides a background signal for comparison with the positively selected pool. We also recommend deep sequencing the initial library in order to facilitate the final in silico analysis of enrichment with respect to the initial levels. Counter selection procedures, using modified or inactive baits, allow strengthening the binding specificity of the winning selected sequences.

  15. Algorithm of detecting structural variations in DNA sequences

    Science.gov (United States)

    Nałecz-Charkiewicz, Katarzyna; Nowak, Robert

    2014-11-01

    Whole genome sequencing enables to use the longest common subsequence algorithm to detect genetic structure variations. We propose to search position of short unique fragments, genetic markers, to achieve acceptable time and space complexity. The markers are generated by algorithms searching the genetic sequence or its Fourier transformation. The presented methods are checked on structural variations generated in silico on bacterial genomes giving the comparable or better results than other solutions.

  16. An improved SELEX technique for selection of DNA aptamers binding to M-type 11 of Streptococcus pyogenes.

    Science.gov (United States)

    Hamula, Camille L A; Peng, Hanyong; Wang, Zhixin; Tyrrell, Gregory J; Li, Xing-Fang; Le, X Chris

    2016-03-15

    Streptococcus pyogenes is a clinically important pathogen consisting of various serotypes determined by different M proteins expressed on the cell surface. The M type is therefore a useful marker to monitor the spread of invasive S. pyogenes in a population. Serotyping and nucleic acid amplification/sequencing methods for the identification of M types are laborious, inconsistent, and usually confined to reference laboratories. The primary objective of this work is to develop a technique that enables generation of aptamers binding to specific M-types of S. pyogenes. We describe here an in vitro technique that directly used live bacterial cells and the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) strategy. Live S. pyogenes cells were incubated with DNA libraries consisting of 40-nucleotides randomized sequences. Those sequences that bound to the cells were separated, amplified using polymerase chain reaction (PCR), purified using gel electrophoresis, and served as the input DNA pool for the next round of SELEX selection. A specially designed forward primer containing extended polyA20/5Sp9 facilitated gel electrophoresis purification of ssDNA after PCR amplification. A counter-selection step using non-target cells was introduced to improve selectivity. DNA libraries of different starting sequence diversity (10(16) and 10(14)) were compared. Aptamer pools from each round of selection were tested for their binding to the target and non-target cells using flow cytometry. Selected aptamer pools were then cloned and sequenced. Individual aptamer sequences were screened on the basis of their binding to the 10 M-types that were used as targets. Aptamer pools obtained from SELEX rounds 5-8 showed high affinity to the target S. pyogenes cells. Tests against non-target Streptococcus bovis, Streptococcus pneumoniae, and Enterococcus species demonstrated selectivity of these aptamers for binding to S. pyogenes. Several aptamer sequences were found to bind

  17. Bioactivity of 2′-deoxyinosine-incorporated aptamer AS1411

    Science.gov (United States)

    Fan, Xinmeng; Sun, Lidan; Wu, Yun; Zhang, Lihe; Yang, Zhenjun

    2016-01-01

    Aptamers can be chemically modified to enhance nuclease resistance and increase target affinity. In this study, we performed chemical modification of 2′-deoxyinosine in AS1411, an anti-proliferative G-rich oligodeoxynucleotide aptamer, which binds selectively to the nucleolin protein. Its function was augmented when 2′-deoxyinosine was incorporated at positions 12, 13, 15, and 24 of AS1411, respectively. In addition, double incorporation of 2′-deoxyinosine at positions 12 and 24 (FAN-1224dI), 13 and 24 (FAN-1324dI), and 15 and 24 (FAN-1524dI) promoted G-quartet formation, as well as inhibition of DNA replication and tumor cell growth, and induced S-phase cell cycle arrest. In further animal experiments, FAN-1224dI, FAN-1324dI and FAN-1524dI resulted in enhanced treatment effects than AS1411 alone. These results suggested that the position and number of modification substituents in AS1411 are critical parameters to improve the diagnostic and therapeutic function of the aptamer. Structural investigations of the FAN-1524dI/nucleolin complex structure, using molecular dynamics simulation, revealed the critical interactions involving nucleolin and 2′-dI incorporated AS1411 compared with AS1411 alone. These findings augment understanding of the role of 2′-deoxyinosine moieties in interactive binding processes. PMID:27194215

  18. Bioactivity of 2'-deoxyinosine-incorporated aptamer AS1411.

    Science.gov (United States)

    Fan, Xinmeng; Sun, Lidan; Wu, Yun; Zhang, Lihe; Yang, Zhenjun

    2016-05-19

    Aptamers can be chemically modified to enhance nuclease resistance and increase target affinity. In this study, we performed chemical modification of 2'-deoxyinosine in AS1411, an anti-proliferative G-rich oligodeoxynucleotide aptamer, which binds selectively to the nucleolin protein. Its function was augmented when 2'-deoxyinosine was incorporated at positions 12, 13, 15, and 24 of AS1411, respectively. In addition, double incorporation of 2'-deoxyinosine at positions 12 and 24 (FAN-1224dI), 13 and 24 (FAN-1324dI), and 15 and 24 (FAN-1524dI) promoted G-quartet formation, as well as inhibition of DNA replication and tumor cell growth, and induced S-phase cell cycle arrest. In further animal experiments, FAN-1224dI, FAN-1324dI and FAN-1524dI resulted in enhanced treatment effects than AS1411 alone. These results suggested that the position and number of modification substituents in AS1411 are critical parameters to improve the diagnostic and therapeutic function of the aptamer. Structural investigations of the FAN-1524dI/nucleolin complex structure, using molecular dynamics simulation, revealed the critical interactions involving nucleolin and 2'-dI incorporated AS1411 compared with AS1411 alone. These findings augment understanding of the role of 2'-deoxyinosine moieties in interactive binding processes.

  19. Accuracy of structure-based sequence alignment of automatic methods

    Directory of Open Access Journals (Sweden)

    Lee Byungkook

    2007-09-01

    Full Text Available Abstract Background Accurate sequence alignments are essential for homology searches and for building three-dimensional structural models of proteins. Since structure is better conserved than sequence, structure alignments have been used to guide sequence alignments and are commonly used as the gold standard for sequence alignment evaluation. Nonetheless, as far as we know, there is no report of a systematic evaluation of pairwise structure alignment programs in terms of the sequence alignment accuracy. Results In this study, we evaluate CE, DaliLite, FAST, LOCK2, MATRAS, SHEBA and VAST in terms of the accuracy of the sequence alignments they produce, using sequence alignments from NCBI's human-curated Conserved Domain Database (CDD as the standard of truth. We find that 4 to 9% of the residues on average are either not aligned or aligned with more than 8 residues of shift error and that an additional 6 to 14% of residues on average are misaligned by 1–8 residues, depending on the program and the data set used. The fraction of correctly aligned residues generally decreases as the sequence similarity decreases or as the RMSD between the Cα positions of the two structures increases. It varies significantly across CDD superfamilies whether shift error is allowed or not. Also, alignments with different shift errors occur between proteins within the same CDD superfamily, leading to inconsistent alignments between superfamily members. In general, residue pairs that are more than 3.0 Å apart in the reference alignment are heavily (>= 25% on average misaligned in the test alignments. In addition, each method shows a different pattern of relative weaknesses for different SCOP classes. CE gives relatively poor results for β-sheet-containing structures (all-β, α/β, and α+β classes, DaliLite for "others" class where all but the major four classes are combined, and LOCK2 and VAST for all-β and "others" classes. Conclusion When the sequence

  20. Sequence structure of Lowary/Widom clones forming strong nucleosomes.

    Science.gov (United States)

    Trifonov, Edward N

    2016-01-01

    Lowary and Widom selected from random sequences those which form exceptionally stable nucleosomes, including clone 601, the current champion of strong nucleosome (SN) sequences. This unique sequence database (LW sequences) carries sequence elements which confer stability on the nucleosomes formed on the sequences, and, thus, may serve as source of information on the structure of "ideal" or close to ideal nucleosome DNA sequence. An important clue is also provided by crystallographic study of Vasudevan and coauthors on clone 601 nucleosomes. It demonstrated that YR·YR dinucleotide stacks (primarily TA·TA) follow one another at distances 10 or 11 bases or multiples thereof, such that they all are located on the interface between DNA and histone octamer. Combining this important information with alignment of the YR-containing 10-mers and 11-mers from LW sequences, the bendability matrices of the stable nucleosome DNA are derived. The matrices suggest that the periodically repeated TA (YR), RR, and YY dinucleotides are the main sequence features of the SNs. This consensus coincides with the one for recently discovered SNs with visibly periodic DNA sequences. Thus, the experimentally observed stable LW nucleosomes and SNs derived computationally appear to represent the same entity - exceptionally stable SNs.

  1. Design of Aptamer-Based Detector for Trinitrotoluene (TNT and Review of Its Performance

    Directory of Open Access Journals (Sweden)

    Ramin Torshizi

    2016-06-01

    Full Text Available Background: The aim of this work is to achieve a DNA sequence of the aptamer that is specifically able to detect the trinitrotoluene (TNT. Materials and Methods: Amplification of nucleotide fragments (PCR was performed using special primers and a library of random nucleotides (two determined sequences of 19 and 21 nucleotides forconnecting to primers and 78 randomized nucleotides in the center. In order to perform SELEX1 study using magnetic nanoparticles and the EDC2 reagent, TNT3 as binds to albumin (TNP-BSA was immobilized. After initial screening, PCR were repeated on isolated pieces of nanoparticles using digoxigenin (DIG-labeled nucleotides. In order to evaluate the aptamer function, using ELONA4 technique (Enzyme Linked Oligonucleotide Assay and a specific anti-DIG antibody which is conjugated to peroxidase, the performance of aptamer for TNT detection was studied. Finally, this sequence is cloned into pBluescript plasmid and sequenced. Results and Conclusion: The cloned aptamer has good efficiency for detection of TNT and could be used as aptasensor for detection of TNT in future studies.

  2. A benchmark of multiple sequence alignment programs upon structural RNAs

    DEFF Research Database (Denmark)

    Gardner, P. P.; Wilm, A.; Washietl, S.

    2005-01-01

    , we systematically test the performance of existing alignment algorithms on structural RNAs. This work was aimed at achieving the following goals: (i) to determine conditions where it is appropriate to apply common sequence alignment methods to the structural RNA alignment problem. This indicates......To date, few attempts have been made to benchmark the alignment algorithms upon nucleic acid sequences. Frequently, sophisticated PAM or BLOSUM like models are used to align proteins, yet equivalents are not considered for nucleic acids; instead, rather ad hoc models are generally favoured. Here...... where and when researchers should consider augmenting the alignment process with auxiliary information, such as secondary structure and (ii) to determine which sequence alignment algorithms perform well under the broadest range of conditions. We find that sequence alignment alone, using the current...

  3. Aptamer Selection Express: A Novel Method for Rapid Single-Step Selection and Sensing of Aptamers

    Science.gov (United States)

    2008-12-01

    aptamers of Bacillus anthracis (Ba), Shiga toxin , botulinum neurotoxin (BoNT), and Francisella tularensis bacteria (all selected by SELEX) have been...This process has been used to select aptamers against different types of targets ( Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2...studied by reselecting aptamers against different targets, Ba spores, Shiga toxin , and F. tularensis bacteria. In contrast to SELEX, the use of

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

    Science.gov (United States)

    Szeto, Kylan; Craighead, Harold G.

    2014-09-01

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

  5. Screening and characterization of aptamers of Cε3-Cε4 protein%Cε3-Cε4蛋白核酸适配子筛选与鉴定

    Institute of Scientific and Technical Information of China (English)

    刘中成; 赵丽君; 张艳芬; 时海浪; 解瑶

    2012-01-01

    In order to obtain nucleotides aptamers bind to IgE, 80 bp nucleotides single-stranded DNA library containing 40 random nucleotides was designed and synthesized. Oligonucleotides that bind to human Cε3— Cε4 protein were isolated from ssDNA pools by the systematic evolution of ligands by exponential enrichment (SELEX) method using nitrocellulose filters as screening medium. Through the optimization of critical PCR and asymmetric PCR parameters including annealing temperature, cycles, and molar ratios of target protein and ssDNA etc, a suitable screening system was established. The aptamers of Cε3-Cε4 protein with high affinity and high specificity were identified by ELISA with biotin-streptavidin-horseradish peroxidase system, and its primary sequence and second structure were analyzed by DNAMAN package and DNA folding sever after being cloned and sequenced. Moreover, target protein was bound to one aptamer and another aptamer modified with biotion together forming a sandwich-like complex, which was captured in microwell to detect IgE concentration using the optimal combination in the sandwich method named enzyme-linked aptamers sorption assay (ELASA). The method could be used for the quantitative detection of human IgE, and whose sensitivity reached to 120 ng·mL-1.%设计并合成随机ssDNA文库,以硝酸纤维素膜为筛选介质,利用SELEX技术筛选人Cε3 -Cε4蛋白特异性高亲和力核酸适配子,通过对退火温度、循环次数及靶蛋白与ssDNA摩尔比等关键参数进行优化,建立了适合的筛选体系.ELISA测定各适配子亲和力,获得了人Cε3 -Cε4蛋白高亲和力、高特异性适配子,并分析了其一级结构和二级结构.以所获高亲和力适配子分别作为捕获适配子和检测适配子,建立了酶联适配子吸附试验(enzyme-linked aptamers sorption assay,ELASA)方法,灵敏度达到120 ng.mL-l,可用于人IgE的定量检测.

  6. High performance aptamer affinity chromatography for single-step selective extraction and screening of basic protein lysozyme.

    Science.gov (United States)

    Han, Bin; Zhao, Chao; Yin, Junfa; Wang, Hailin

    2012-08-15

    A DNA aptamer based high-performance affinity chromatography is developed for selective extraction and screening of a basic protein lysozyme. First, a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic column was synthesized in situ by thermally initiated radical polymerization, and then an anti-lysozyme DNA aptamer was covalently immobilized on the surface of the monolith through a 16-atom spacer arm. The target protein lysozyme but non-target proteins can be trapped by the immobilized anti-lysozyme DNA aptamer. In contrast, lysozyme cannot be trapped by the immobilized oligodeoxynucleotide that does not contain the sequence of the anti-lysozyme DNA aptamer. The study clearly demonstrates the trapping of lysozyme by the immobilized anti-lysozyme DNA aptamer is mainly due to specific recognition rather than simple electrostatic interaction of positively charged protein and the negatively charged DNA. The inter-day precision was determined as 0.8% for migration time and 4.2% for peak area, respectively. By the use of aptamer affinity monolith, a screening strategy is developed to selectively extract lysozyme from chicken egg white, showing the advantages of high efficiency, low cost and ease-of-operation.

  7. Cell-Specific Aptamers as Emerging Therapeutics

    Directory of Open Access Journals (Sweden)

    Cindy Meyer

    2011-01-01

    Full Text Available Aptamers are short nucleic acids that bind to defined targets with high affinity and specificity. The first aptamers have been selected about two decades ago by an in vitro process named SELEX (systematic evolution of ligands by exponential enrichment. Since then, numerous aptamers with specificities for a variety of targets from small molecules to proteins or even whole cells have been selected. Their applications range from biosensing and diagnostics to therapy and target-oriented drug delivery. More recently, selections using complex targets such as live cells have become feasible. This paper summarizes progress in cell-SELEX techniques and highlights recent developments, particularly in the field of medically relevant aptamers with a focus on therapeutic and drug-delivery applications.

  8. Informational structure of genetic sequences and nature of gene splicing

    Science.gov (United States)

    Trifonov, E. N.

    1991-10-01

    Only about 1/20 of DNA of higher organisms codes for proteins, by means of classical triplet code. The rest of DNA sequences is largely silent, with unclear functions, if any. The triplet code is not the only code (message) carried by the sequences. There are three levels of molecular communication, where the same sequence ``talks'' to various bimolecules, while having, respectively, three different appearances: DNA, RNA and protein. Since the molecular structures and, hence, sequence specific preferences of these are substantially different, the original DNA sequence has to carry simultaneously three types of sequence patterns (codes, messages), thus, being a composite structure in which one had the same letter (nucleotide) is frequently involved in several overlapping codes of different nature. This multiplicity and overlapping of the codes is a unique feature of the Gnomic, language of genetic sequences. The coexisting codes have to be degenerate in various degrees to allow an optimal and concerted performance of all the encoded functions. There is an obvious conflict between the best possible performance of a given function and necessity to compromise the quality of a given sequence pattern in favor of other patterns. It appears that the major role of various changes in the sequences on their ``ontogenetic'' way from DNA to RNA to protein, like RNA editing and splicing, or protein post-translational modifications is to resolve such conflicts. New data are presented strongly indicating that the gene splicing is such a device to resolve the conflict between the code of DNA folding in chromatin and the triplet code for protein synthesis.

  9. Selection, characterization and application of nucleic acid aptamers for the capture and detection of human norovirus strains.

    Directory of Open Access Journals (Sweden)

    Blanca I Escudero-Abarca

    Full Text Available Human noroviruses (HuNoV are the leading cause of acute viral gastroenteritis and an important cause of foodborne disease. Despite their public health significance, routine detection of HuNoV in community settings, or food and environmental samples, is limited, and there is a need to develop alternative HuNoV diagnostic reagents to complement existing ones. The purpose of this study was to select and characterize single-stranded (ssDNA aptamers with binding affinity to HuNoV. The utility of these aptamers was demonstrated in their use for capture and detection of HuNoV in outbreak-derived fecal samples and a representative food matrix. SELEX (Systematic Evolution of Ligands by EXponential enrichment was used to isolate ssDNA aptamer sequences with broad reactivity to the prototype GII.2 HuNoV strain, Snow Mountain Virus (SMV. Four aptamer candidates (designated 19, 21, 25 and 26 were identified and screened for binding affinity to 14 different virus-like particles (VLPs corresponding to various GI and GII HuNoV strains using an Enzyme-Linked Aptamer Sorbant Assay (ELASA. Collectively, aptamers 21 and 25 showed affinity to 13 of the 14 VLPs tested, with strongest binding to GII.2 (SMV and GII.4 VLPs. Aptamer 25 was chosen for further study. Its binding affinity to SMV-VLPs was equivalent to that of a commercial antibody within a range of 1 to 5 µg/ml. Aptamer 25 also showed binding to representative HuNoV strains present in stool specimens obtained from naturally infected individuals. Lastly, an aptamer magnetic capture (AMC method using aptamer 25 coupled with RT-qPCR was developed for recovery and detection of HuNoV in artificially contaminated lettuce. The capture efficiency of the AMC was 2.5-36% with an assay detection limit of 10 RNA copies per lettuce sample. These ssDNA aptamer candidates show promise as broadly reactive reagents for use in HuNoV capture and detection assays in various sample types.

  10. Feasible Study of Vitro Selection of DNA Aptamers against Streptococcus Mutans UA159 by SELEX%SELEX技术筛选变形链球菌UA159适配子可行性的研究

    Institute of Scientific and Technical Information of China (English)

    黄博; 彭磊; 王帅; 刘筱娣; 郭丽宏

    2012-01-01

    Objective: To verify the feasibility of vitro selection of DNA aptamers against Streptococcus mutaris UA159 by SELEX. Methods: Streptococcus Mutans UA159, Lactobacilli and 1.5mL centrifuge Tube are chosen to be target materials. And in vitro synthesized 35 mer random DNA library (101213 types of different DNAs) is subjected to selection using SELEX method against Streptococcus Mutans UA159, Lactobacilli and 1.5mL centrifuge Tube. The selected aptamers are amplified by asymmetric PCR, then cloned and sequenced. Finally, the structures of selected aptamers are analyzed. Results: The structure showed specific secondary structure shape characterized by their loops, bulges and hairpins in the second round. Conclusion: This sutdy shows the SELEX has the potential to be used as a selection tool to acquire aptamers with high affinity and specificity against oral bacteria.%目的:研究SELEX技术用于筛选口腔致龋菌适配子的可行性.方法:化学合成长度为35mer的随机ssDNA文库,利用SELEX技术,分别以变形链球菌UA159(以下简称变链UA159)、乳杆菌和离心管作为靶物质,筛选适配子,不对称PCR扩增筛选产物,所得适配子进行克隆、测序,分析其二级结构,并对其二级结构进行了初步分析.结果:显示各个靶物质的筛选产物在第二轮筛选时就已经表现出具有特征性的二级结构.结论:SELEX技术可以用于口腔致龋菌适配子的筛选.

  11. Protein 3D structure computed from evolutionary sequence variation.

    Directory of Open Access Journals (Sweden)

    Debora S Marks

    Full Text Available The evolutionary trajectory of a protein through sequence space is constrained by its function. Collections of sequence homologs record the outcomes of millions of evolutionary experiments in which the protein evolves according to these constraints. Deciphering the evolutionary record held in these sequences and exploiting it for predictive and engineering purposes presents a formidable challenge. The potential benefit of solving this challenge is amplified by the advent of inexpensive high-throughput genomic sequencing.In this paper we ask whether we can infer evolutionary constraints from a set of sequence homologs of a protein. The challenge is to distinguish true co-evolution couplings from the noisy set of observed correlations. We address this challenge using a maximum entropy model of the protein sequence, constrained by the statistics of the multiple sequence alignment, to infer residue pair couplings. Surprisingly, we find that the strength of these inferred couplings is an excellent predictor of residue-residue proximity in folded structures. Indeed, the top-scoring residue couplings are sufficiently accurate and well-distributed to define the 3D protein fold with remarkable accuracy.We quantify this observation by computing, from sequence alone, all-atom 3D structures of fifteen test proteins from different fold classes, ranging in size from 50 to 260 residues, including a G-protein coupled receptor. These blinded inferences are de novo, i.e., they do not use homology modeling or sequence-similar fragments from known structures. The co-evolution signals provide sufficient information to determine accurate 3D protein structure to 2.7-4.8 Å C(α-RMSD error relative to the observed structure, over at least two-thirds of the protein (method called EVfold, details at http://EVfold.org. This discovery provides insight into essential interactions constraining protein evolution and will facilitate a comprehensive survey of the universe of

  12. Aptamers provide superior stainings of cellular receptors studied under super-resolution microscopy

    Science.gov (United States)

    Höbartner, Claudia

    2017-01-01

    Continuous improvements in imaging techniques are challenging biologists to search for more accurate methods to label cellular elements. This is particularly relevant for diffraction-unlimited fluorescence imaging, where the perceived resolution is affected by the size of the affinity probes. This is evident when antibodies, which are 10–15 nm in size, are used. Previously it has been suggested that RNA aptamers (~3 nm) can be used to detect cellular proteins under super-resolution imaging. However, a direct comparison between several aptamers and antibodies is needed, to clearly show the advantages and/or disadvantages of the different probes. Here we have conducted such a comparative study, by testing several aptamers and antibodies using stimulated emission depletion microscopy (STED). We have targeted three membrane receptors, EGFR, ErbB2 and Epha2, which are relevant to human health, and recycle between plasma membrane and intracellular organelles. Our results suggest that the aptamers can reveal more epitopes than most antibodies, thus providing a denser labeling of the stained structures. Moreover, this improves the overall quality of the information that can be extracted from the images. We conclude that aptamers could become useful fluorescent labeling tools for light microscopy and super-resolution imaging, and that their development for novel targets is imperative. PMID:28235049

  13. Prostate-specific RNA aptamer: promising nucleic acid antibody-like cancer detection.

    Science.gov (United States)

    Marangoni, Karina; Neves, Adriana F; Rocha, Rafael M; Faria, Paulo R; Alves, Patrícia T; Souza, Aline G; Fujimura, Patrícia T; Santos, Fabiana A A; Araújo, Thaise G; Ward, Laura S; Goulart, Luiz R

    2015-07-15

    We described the selection of a novel nucleic acid antibody-like prostate cancer (PCa) that specifically binds to the single-stranded DNA molecule from a 277-nt fragment that may have been partially paired and bound to the PCA3 RNA conformational structure. PCA3-277 aptamer ligands were obtained, and the best binding molecule, named CG3, was synthesized for validation. Aiming to prove its diagnostic utility, we used an apta-qPCR assay with CG3-aptamer conjugated to magnetic beads to capture PCA3 transcripts, which were amplified 97-fold and 7-fold higher than conventional qPCR in blood and tissue, respectively. Histopathologic analysis of 161 prostate biopsies arranged in a TMA and marked with biotin-labeled CG3-aptamer showed moderate staining in both cytoplasm and nucleus of PCa samples; in contrast, benign prostatic hyperplasia (BPH) samples presented strong nuclear staining (78% of the cases). No staining was observed in stromal cells. In addition, using an apta-qPCR, we demonstrated that CG3-aptamer specifically recognizes the conformational PCA3-277 molecule and at least three other transcript variants, indicating that long non-coding RNA (lncRNA) is processed after transcription. We suggest that CG3-aptamer may be a useful PCa diagnostic tool. In addition, this molecule may be used in drug design and drug delivery for PCa therapy.

  14. Code domains in tandem repetitive DNA sequence structures.

    Science.gov (United States)

    Vogt, P

    1992-10-01

    Traditionally, many people doing research in molecular biology attribute coding properties to a given DNA sequence if this sequence contains an open reading frame for translation into a sequence of amino acids. This protein coding capability of DNA was detected about 30 years ago. The underlying genetic code is highly conserved and present in every biological species studied so far. Today, it is obvious that DNA has a much larger coding potential for other important tasks. Apart from coding for specific RNA molecules such as rRNA, snRNA and tRNA molecules, specific structural and sequence patterns of the DNA chain itself express distinct codes for the regulation and expression of its genetic activity. A chromatin code has been defined for phasing of the histone-octamer protein complex in the nucleosome. A translation frame code has been shown to exist that determines correct triplet counting at the ribosome during protein synthesis. A loop code seems to organize the single stranded interaction of the nascent RNA chain with proteins during the splicing process, and a splicing code phases successive 5' and 3' splicing sites. Most of these DNA codes are not exclusively based on the primary DNA sequence itself, but also seem to include specific features of the corresponding higher order structures. Based on the view that these various DNA codes are genetically instructive for specific molecular interactions or processes, important in the nucleus during interphase and during cell division, the coding capability of tandem repetitive DNA sequences has recently been reconsidered.

  15. The dual aptamer approach: rational design of a high-affinity FAD aptamer.

    Science.gov (United States)

    Merkle, T; Holder, I T; Hartig, J S

    2016-01-14

    A design strategy for high-affinity aptamers of complex biomolecules is presented. We developed an RNA with FAD-binding properties by combining known ATP- and FMN-aptamers. Cooperative binding of FAD was shown by SPR spectroscopy and fluorescence assays. The strategy should be transferable to several other biomolecules.

  16. Label-free triple-helix aptamer as sensing platform for "signal-on" fluorescent detection of thrombin.

    Science.gov (United States)

    Xu, Nan; Wang, Quanbo; Lei, Jianping; Liu, Lin; Ju, Huangxian

    2015-01-01

    The design of a label-free aptamer for separation of recognition sequence from signal reporter is significant to ensure the high-efficiency affinity between aptamer and target. This work develops a label-free triple-helix aptamer (THA) as sensing platform for "signal-on" fluorescent detection of thrombin. THA was composed of aptamer sequence and help DNA 1 (H1), which contained the complementary sequence of hexachloro-fluorescein (HEX) labeled help DNA 2 (H2). The specific recognition event between aptamer and thrombin triggered the dismission of THA to release H1. The released H1 then reacted with the signal probe of H2/graphene oxide (GO) nanocomposite to form H1-H2 duplex, leading to the fluorescence recovery of H2 due to the detachment of H1-H2 duplex from the surface of GO. With employment of THA as a signal transducer and GO as a "superquencher", this method shows a sensitive response to thrombin with a wide concentration range from 5 to 1200 nM. The limit of detection is 1.8 nM (S/N=3) with excellent selectivity. Considering the universality of THA, the proposed aptasensor would provide a platform for homogeneous fluorescent detection of a wide range of analytes.

  17. Generation of Internal-Image Functional Aptamers of Okadaic Acid via Magnetic-Bead SELEX

    Directory of Open Access Journals (Sweden)

    Chao Lin

    2015-12-01

    Full Text Available Okadaic acid (OA is produced by Dinophysis and Prorocentrum dinoflagellates and primarily accumulates in bivalves, and this toxin has harmful effects on consumers and operators. In this work, we first report the use of aptamers as novel non-toxic probes capable of binding to a monoclonal antibody against OA (OA-mAb. Aptamers that mimic the OA toxin with high affinity and selectivity were generated by the magnetic bead-assisted systematic evolution of ligands by exponential enrichment (SELEX strategy. After 12 selection rounds, cloning, sequencing and enzyme-linked immunosorbent assay (ELISA analysis, four candidate aptamers (O24, O31, O39, O40 were selected that showed high affinity and specificity for OA-mAb. The affinity constants of O24, O31, O39 and O40 were 8.3 × 108 M−1, 1.47 × 109 M−1, 1.23 × 109 M−1 and 1.05 × 109 M−1, respectively. Indirect competitive ELISA was employed to determine the internal-image function of the aptamers. The results reveal that O31 has a similar competitive function as free OA toxin, whereas the other three aptamers did not bear the necessary internal-image function. Based on the derivation of the curvilinear equation for OA/O31, the equation that defined the relationship between the OA toxin content and O31 was Y = 2.185X − 1.78. The IC50 of O31 was 3.39 ng·mL−1, which was close to the value predicted by the OA ELISA (IC50 = 4.4 ng·mL−1; the IC10 was 0.33 ng·mL−1. The above data provides strong evidence that internal-image functional aptamers could be applicable as novel probes in a non-toxic assay.

  18. The aptamer DNA-templated fluorescence silver nanoclusters: ATP detection and preliminary mechanism investigation.

    Science.gov (United States)

    Xu, Jiaona; Wei, Chunying

    2017-01-15

    Two general and reliable fluorescence sensors were proposed in this work utilizing aptamer DNA-templated silver nanoclusters (Ag NCs). Both DNA-AgNCs could be used for label-free detecting of ATP with the limits of detection of 0.44 and 0.65mM. One of them was further applied to monitor the activity of adenosine deaminase (ADA). In our effort to elucidate the light-up mechanism, we studied a total of six Ag NCs prepared by different DNA sequences, and found that they showed different sensitivity to ATP. Both BT3T3- and BT3T3(R)-templated Ag NCs were chose to make particular studies by UV-vis, TEM, fluorescence, and TCSPC methods. The results showed that when DNA-Ag NCs was kept for 1.5h and presented a strong fluorescence, the addition of ATP failed to cause a large change of fluorescence intensity; on the contrary, after Ag NCs was kept for 24h and emitted a weak fluorescence, adding ATP was able to result in the large fluorescence enhanced of 43 and 33 times for BT3T3- and BT3T3(R)-templated Ag NCs, respectively. The possible mechanism was also suggested that ATP binding to aptamer segment of template induced the change of the DNA secondary structure, which made the aggregated Ag nanoparticles disperse into Ag NCs with an average diameter of about 2nm that were responsible for the large fluorescence increase. Moreover, ATP could protect the fluorescence intensity of BT3T3(R)-templated Ag NCs from quenching for at least 9h.

  19. Raman and surface-enhanced Raman scattering (SERS) studies of the thrombin-binding aptamer.

    Science.gov (United States)

    Wu, Tsai-Chin; Vasudev, Milana; Dutta, Mitra; Stroscio, Michael A

    2013-06-01

    Surface-enhanced Raman scattering is used to study the Raman spectra and peak shifts the thrombin-binding aptamer (TBA) on substrates having two different geometries; one with a single stranded sequence and one with double stranded sequence. The Raman signals of the deoxyribonucleic acids on both substrates are enhanced and specific peaks of bases are identified. These results are highly reproducible and have promising applications in low cost nucleic acid detection.

  20. Arrest of rolling circle amplification by protein-binding DNA aptamers.

    Science.gov (United States)

    Wang, Lida; Tram, Kha; Ali, Monsur M; Salena, Bruno J; Li, Jinghong; Li, Yingfu

    2014-02-24

    Certain DNA polymerases, such as ϕ29 DNA polymerase, can isothermally copy the sequence of a circular template round by round in a process known as rolling circle amplification (RCA), which results in super-long single-stranded (ss) DNA molecules made of tandem repeats. The power of RCA reflects the high processivity and the strand-displacement ability of these polymerases. In this work, the ability of ϕ29DNAP to carry out RCA over circular templates containing a protein-binding DNA aptamer sequence was investigated. It was found that protein-aptamer interactions can prevent this DNA polymerase from reading through the aptameric domain. This finding indicates that protein-binding DNA aptamers can form highly stable complexes with their targets in solution. This novel observation was exploited by translating RCA arrest into a simple and convenient colorimetric assay for the detection of specific protein targets, which continues to showcase the versatility of aptamers as molecular recognition elements for biosensing applications.

  1. Hinge Atlas: relating protein sequence to sites of structural flexibility

    OpenAIRE

    Yang Julie; Lu Long J; Flores Samuel C; Carriero Nicholas; Gerstein Mark B

    2007-01-01

    Abstract Background Relating features of protein sequences to structural hinges is important for identifying domain boundaries, understanding structure-function relationships, and designing flexibility into proteins. Efforts in this field have been hampered by the lack of a proper dataset for studying characteristics of hinges. Results Using the Molecular Motions Database we have created a Hinge Atlas of manually annotated hinges and a statistical formalism for calculating the enrichment of v...

  2. Model identification for DNA sequence-structure relationships.

    Science.gov (United States)

    Hawley, Stephen Dwyer; Chiu, Anita; Chizeck, Howard Jay

    2006-11-01

    We investigate the use of algebraic state-space models for the sequence dependent properties of DNA. By considering the DNA sequence as an input signal, rather than using an all atom physical model, computational efficiency is achieved. A challenge in deriving this type of model is obtaining its structure and estimating its parameters. Here we present two candidate model structures for the sequence dependent structural property Slide and a method of encoding the models so that a recursive least squares algorithm can be applied for parameter estimation. These models are based on the assumption that the value of Slide at a base-step is determined by the surrounding tetranucleotide sequence. The first model takes the four bases individually as inputs and has a median root mean square deviation of 0.90 A. The second model takes the four bases pairwise and has a median root mean square deviation of 0.88 A. These values indicate that the accuracy of these models is within the useful range for structure prediction. Performance is comparable to published predictions of a more physically derived model, at significantly less computational cost.

  3. A Universal Base in a Specific Role: Tuning up a Thrombin Aptamer with 5-Nitroindole

    Science.gov (United States)

    Tsvetkov, Vladimir B.; Varizhuk, Anna M.; Pozmogova, Galina E.; Smirnov, Igor P.; Kolganova, Natalia A.; Timofeev, Edward N.

    2015-11-01

    In this study we describe new modified analogs of the thrombin binding aptamer (TBA) containing 5-nitroindole residues. It has been shown that all modified TBAs form an anti-parallel G-quadruplex structure and retain the ability to inhibit thrombin. The most advanced TBA variant (TBA-N8) has a substantially increased clotting time and two-fold lower IC50 value compared to the unmodified prototype. Molecular modelling studies suggest that the improved anticoagulant properties of TBA-N8 result from changes in the binding mode of the analog. A modified central loop in TBA-N8 is presumed to participate in the binding of the target protein. Studies of FAM labelled TBA and TBA-N8 showed an improved binding affinity of the modified aptamer and provided evidence of a direct interaction between the modified central loop and thrombin. Our findings have implications for the design of new aptamers with improved binding affinities.

  4. Evolution of Complex Target SELEX to Identify Aptamers against Mammalian Cell-Surface Antigens

    Directory of Open Access Journals (Sweden)

    Prabodhika Mallikaratchy

    2017-01-01

    Full Text Available The demand has increased for sophisticated molecular tools with improved detection limits. Such molecules should be simple in structure, yet stable enough for clinical applications. Nucleic acid aptamers (NAAs represent a class of molecules able to meet this demand. In particular, aptamers, a class of small nucleic acid ligands that are composed of single-stranded modified/unmodified RNA/DNA molecules, can be evolved from a complex library using Systematic Evolution of Ligands by EXponential enrichment (SELEX against almost any molecule. Since its introduction in 1990, in stages, SELEX technology has itself undergone several modifications, improving selection and broadening the repertoire of targets. This review summarizes these milestones that have pushed the field forward, allowing researchers to generate aptamers that can potentially be applied as therapeutic and diagnostic agents.

  5. Pairwise local structural alignment of RNA sequences with sequence similarity less than 40%

    DEFF Research Database (Denmark)

    Havgaard, Jakob Hull; Lyngsø, Rune B.; Stormo, Gary D.

    2005-01-01

    Motivation: Searching for non-coding RNA (ncRNA) genes and structural RNA elements (eleRNA) are major challenges in gene finding todya as these often are conserved in structure rather than in sequence. Even though the number of available methods is growing, it is still of interest to pairwise....... The structure prediction performance for a family is typically around 0.7 using Matthews correlation coefficient. In case (2), the algorithm is successful at locating RNA families with an average sensitivity of 0.8 and a positive predictive value of 0.9 using a BLAST-like hit selection scheme. Availability...

  6. Statistical mechanics of secondary structures formed by random RNA sequences.

    Science.gov (United States)

    Bundschuh, R; Hwa, T

    2002-03-01

    The formation of secondary structures by a random RNA sequence is studied as a model system for the sequence-structure problem omnipresent in biopolymers. Several toy energy models are introduced to allow detailed analytical and numerical studies. First, a two-replica calculation is performed. By mapping the two-replica problem to the denaturation of a single homogeneous RNA molecule in six-dimensional embedding space, we show that sequence disorder is perturbatively irrelevant, i.e., an RNA molecule with weak sequence disorder is in a molten phase where many secondary structures with comparable total energy coexist. A numerical study of various models at high temperature reproduces behaviors characteristic of the molten phase. On the other hand, a scaling argument based on the external statistics of rare regions can be constructed to show that the low-temperature phase is unstable to sequence disorder. We performed a detailed numerical study of the low-temperature phase using the droplet theory as a guide, and characterized the statistics of large-scale, low-energy excitations of the secondary structures from the ground state structure. We find the excitation energy to grow very slowly (i.e., logarithmically) with the length scale of the excitation, suggesting the existence of a marginal glass phase. The transition between the low-temperature glass phase and the high-temperature molten phase is also characterized numerically. It is revealed by a change in the coefficient of the logarithmic excitation energy, from being disorder dominated to being entropy dominated.

  7. Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment.

    Science.gov (United States)

    Levay, Agata; Brenneman, Randall; Hoinka, Jan; Sant, David; Cardone, Marco; Trinchieri, Giorgio; Przytycka, Teresa M; Berezhnoy, Alexey

    2015-07-13

    Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs.

  8. Protein Function Prediction Based on Sequence and Structure Information

    KAUST Repository

    Smaili, Fatima Z.

    2016-05-25

    The number of available protein sequences in public databases is increasing exponentially. However, a significant fraction of these sequences lack functional annotation which is essential to our understanding of how biological systems and processes operate. In this master thesis project, we worked on inferring protein functions based on the primary protein sequence. In the approach we follow, 3D models are first constructed using I-TASSER. Functions are then deduced by structurally matching these predicted models, using global and local similarities, through three independent enzyme commission (EC) and gene ontology (GO) function libraries. The method was tested on 250 “hard” proteins, which lack homologous templates in both structure and function libraries. The results show that this method outperforms the conventional prediction methods based on sequence similarity or threading. Additionally, our method could be improved even further by incorporating protein-protein interaction information. Overall, the method we use provides an efficient approach for automated functional annotation of non-homologous proteins, starting from their sequence.

  9. Comparative sequence-structure analysis of Aves insulin.

    Science.gov (United States)

    Islam, Md Mirazul; Aktaruzzaman, M; Mohamed, Zahurin

    2015-01-01

    Normal blood glucose level depends on the availability of insulin and its ability to bind insulin receptor (IR) that regulates the downstream signaling pathway. Insulin sequence and blood glucose level usually vary among animals due to species specificity. The study of genetic variation of insulin, blood glucose level and diabetics symptoms development in Aves is interesting because of its optimal high blood glucose level than mammals. Therefore, it is of interest to study its evolutionary relationship with other mammals using sequence data. Hence, we compiled 32 Aves insulin from GenBank to compare its sequence-structure features with phylogeny for evolutionary inference. The analysis shows long conserved motifs (about 14 residues) for functional inference. These sequences show high leucine content (20%) with high instability index (>40). Amino acid position 11, 14, 16 and 20 are variable that may have contribution to binding to IR. We identified functionally critical variable residues in the dataset for possible genetic implication. Structural models of these sequences were developed for surface analysis towards functional representation. These data find application in the understanding of insulin function across species.

  10. Development of RNA aptamers for detection of Salmonella Enteritidis.

    Science.gov (United States)

    Hyeon, Ji-Yeon; Chon, Jung-Whan; Choi, In-Soo; Park, Chankyu; Kim, Dong-Eun; Seo, Kun-Ho

    2012-04-01

    We developed and evaluated RNA aptamers to analyze their potential for use in detecting Salmonella Enteritidis. The selected aptamer was observed to specifically bind to Salmonella Enteritidis without any cross-reactivity to other Salmonella serovars. Thus, this study suggests that aptamers specific to Salmonella Enteritidis have a high potential for use in presumptive presumptive screening methods or alternative serotyping methods.

  11. Biophysical and structural considerations for protein sequence evolution

    Directory of Open Access Journals (Sweden)

    Grahnen Johan A

    2011-12-01

    Full Text Available Abstract Background Protein sequence evolution is constrained by the biophysics of folding and function, causing interdependence between interacting sites in the sequence. However, current site-independent models of sequence evolutions do not take this into account. Recent attempts to integrate the influence of structure and biophysics into phylogenetic models via statistical/informational approaches have not resulted in expected improvements in model performance. This suggests that further innovations are needed for progress in this field. Results Here we develop a coarse-grained physics-based model of protein folding and binding function, and compare it to a popular informational model. We find that both models violate the assumption of the native sequence being close to a thermodynamic optimum, causing directional selection away from the native state. Sampling and simulation show that the physics-based model is more specific for fold-defining interactions that vary less among residue type. The informational model diffuses further in sequence space with fewer barriers and tends to provide less support for an invariant sites model, although amino acid substitutions are generally conservative. Both approaches produce sequences with natural features like dN/dS Conclusions Simple coarse-grained models of protein folding can describe some natural features of evolving proteins but are currently not accurate enough to use in evolutionary inference. This is partly due to improper packing of the hydrophobic core. We suggest possible improvements on the representation of structure, folding energy, and binding function, as regards both native and non-native conformations, and describe a large number of possible applications for such a model.

  12. Transcriptional pausing coordinates folding of the aptamer domain and the expression platform of a riboswitch.

    Science.gov (United States)

    Perdrizet, George A; Artsimovitch, Irina; Furman, Ran; Sosnick, Tobin R; Pan, Tao

    2012-02-28

    Riboswitches are cis-acting elements that regulate gene expression by affecting transcriptional termination or translational initiation in response to binding of a metabolite. A typical riboswitch is made of an upstream aptamer domain and a downstream expression platform. Both domains participate in the folding and structural rearrangement in the absence or presence of its cognate metabolite. RNA polymerase pausing is a fundamental property of transcription that can influence RNA folding. Here we show that pausing plays an important role in the folding and conformational rearrangement of the Escherichia coli btuB riboswitch during transcription by the E. coli RNA polymerase. This riboswitch consists of an approximately 200 nucleotide, coenzyme B12 binding aptamer domain and an approximately 40 nucleotide expression platform that controls the ribosome access for translational initiation. We found that transcriptional pauses at strategic locations facilitate folding and structural rearrangement of the full-length riboswitch, but have minimal effect on the folding of the isolated aptamer domain. Pausing at these regulatory sites blocks the formation of alternate structures and plays a chaperoning role that couples folding of the aptamer domain and the expression platform. Pausing at strategic locations may be a general mechanism for coordinated folding and conformational rearrangements of riboswitch structures that underlie their response to environmental cues.

  13. Optical Aptamer Probes of Fluorescent Imaging to Rapid Monitoring of Circulating Tumor Cell

    Directory of Open Access Journals (Sweden)

    Ji Yeon Hwang

    2016-11-01

    Full Text Available Fluorescence detecting of exogenous EpCAM (epithelial cell adhesion molecule or muc1 (mucin1 expression correlated to cancer metastasis using nanoparticles provides pivotal information on CTC (circulating tumor cell occurrence in a noninvasive tool. In this study, we study a new skill to detect extracellular EpCAM/muc1 using quantum dot-based aptamer beacon (QD-EpCAM/muc1 ALB (aptamer linker beacon. The QD-EpCAM/muc1 ALB was designed using QDs (quantum dots and probe. The EpCAM/muc1-targeting aptamer contains a Ep-CAM/muc1 binding sequence and BHQ1 (black hole quencher 1 or BHQ2 (black hole quencher2. In the absence of target EpCAM/muc1, the QD-EpCAM/muc1 ALB forms a partial duplex loop-like aptamer beacon and remained in quenched state because the BHQ1/2 quenches the fluorescence signal-on of the QD-EpCAM/muc1 ALB. The binding of EpCAM/muc1 of CTC to the EpCAM/muc1 binding aptamer sequence of the EpCAM/muc1-targeting oligonucleotide triggered the dissociation of the BHQ1/2 quencher and subsequent signal-on of a green/red fluorescence signal. Furthermore, acute inflammation was stimulated by trigger such as caerulein in vivo, which resulted in increased fluorescent signal of the cy5.5-EpCAM/muc1 ALB during cancer metastasis due to exogenous expression of EpCAM/muc1 in Panc02-implanted mouse model.

  14. The structure of verbal sequences analyzed with unsupervised learning techniques

    CERN Document Server

    Recanati, Catherine; Bennani, Younès

    2007-01-01

    Data mining allows the exploration of sequences of phenomena, whereas one usually tends to focus on isolated phenomena or on the relation between two phenomena. It offers invaluable tools for theoretical analyses and exploration of the structure of sentences, texts, dialogues, and speech. We report here the results of an attempt at using it for inspecting sequences of verbs from French accounts of road accidents. This analysis comes from an original approach of unsupervised training allowing the discovery of the structure of sequential data. The entries of the analyzer were only made of the verbs appearing in the sentences. It provided a classification of the links between two successive verbs into four distinct clusters, allowing thus text segmentation. We give here an interpretation of these clusters by applying a statistical analysis to independent semantic annotations.

  15. Incorporating secondary structural features into sequence information for predicting protein structural class.

    Science.gov (United States)

    Liao, Bo; Peng, Ting; Chen, Haowen; Lin, Yaping

    2013-10-01

    Knowledge of structural classes is applied in numerous important predictive tasks that address structural and functional features of proteins, although the prediction accuracy of the protein structural classes is not high. In this study, 45 different features were rationally designed to model the differences between protein structural classes, among which, 30 of them reflect the combined protein sequence information. In terms of correlation function, the protein sequence can be converted to a digital signal sequence, from which we can generate 20 discrete Fourier spectrum numbers. According to the segments of amino with different characteristics occurring in protein sequences, the frequencies of the 10 kinds of segments of amino acid (motifs) in protein are calculated. Other features include the secondary structural information :10 features were proposed to model the strong adjacent correlations in the secondary structural elements and capture the long-range spatial interactions between secondary structures, other 5 features were designed to differentiate α/β from α+β classes , which is a major problem of the existing algorithm. The methods were proposed based on a large set of low-identity sequences for which secondary structure is predicted from their sequence (based on PSI-PRED). By means of this method, the overall prediction accuracy of four benchmark datasets were all improved. Especially for the dataset FC699, 25PDB and D1189 which are 1.26%, 1% and 0.85% higher than the best previous method respectively.

  16. Sequence analysis and structural implications of rotavirus capsid proteins.

    Science.gov (United States)

    Parbhoo, N; Dewar, J B; Gildenhuys, S

    Rotavirus is the major cause of severe virus-associated gastroenteritis worldwide in children aged 5 and younger. Many children lose their lives annually due to this infection and the impact is particularly pronounced in developing countries. The mature rotavirus is a non-enveloped triple-layered nucleocapsid containing 11 double stranded RNA segments. Here a global view on the sequence and structure of the three main capsid proteins, VP2, VP6 and VP7 is shown by generating a consensus sequence for each of these rotavirus proteins, for each species obtained from published data of representative rotavirus genotypes from across the world and across species. Degree of conservation between species was represented on homology models for each of the proteins. VP7 shows the highest level of variation with 14-45 amino acids showing conservation of less than 60%. These changes are localised to the outer surface alluding to a possible mechanism in evading the immune system. The middle layer, VP6 shows lower variability with only 14-32 sites having lower than 70% conservation. The inner structural layer made up of VP2 showed the lowest variability with only 1-16 sites having less than 70% conservation across species. The results correlate with each protein's multiple structural roles in the infection cycle. Thus, although the nucleotide sequences vary due to the error-prone nature of replication and lack of proof reading, the corresponding amino acid sequence of VP2, 6 and 7 remain relatively conserved. Benefits of this knowledge about the conservation include the ability to target proteins at sites that cannot undergo mutational changes without influencing viral fitness; as well as possibility to study systems that are highly evolved for structure and function in order to determine how to generate and manipulate such systems for use in various biotechnological applications.

  17. Aptamer-Based Molecular Recognition of Lysergamine, Metergoline and Small Ergot Alkaloids

    Directory of Open Access Journals (Sweden)

    Johan Robbens

    2012-12-01

    Full Text Available Ergot alkaloids are mycotoxins produced by fungi of the genus Claviceps, which infect cereal crops and grasses. The uptake of ergot alkaloid contaminated cereal products can be lethal to humans and animals. For food safety assessment, analytical techniques are currently used to determine the presence of ergot alkaloids in food and feed samples. However, the number of samples which can be analyzed is limited, due to the cost of the equipment and the need for skilled personnel. In order to compensate for the lack of rapid tests for the detection of ergot alkaloids, the aim of this study was to develop a specific recognition element for ergot alkaloids, which could be further applied to produce a colorimetric reaction in the presence of these toxins. As recognition elements, single-stranded DNA ligands were selected by using an iterative selection procedure named SELEX, i.e., Systematic Evolution of Ligands by EXponential enrichment. After several selection cycles, the resulting aptamers were cloned and sequenced. A surface plasmon resonance analysis enabled determination of the dissociation constants of the complexes of aptamers and lysergamine. Dissociation constants in the nanomolar range were obtained with three selected aptamers. One of the selected aptamers, having a dissociation constant of 44 nM, was linked to gold nanoparticles and it was possible to produce a colorimetric reaction in the presence of lysergamine. This system could also be applied to small ergot alkaloids in an ergot contaminated flour sample.

  18. Aptamer nanomedicine for cancer therapeutics: barriers and potential for translation.

    Science.gov (United States)

    Lao, Yeh-Hsing; Phua, Kyle K L; Leong, Kam W

    2015-03-24

    Aptamer nanomedicine, including therapeutic aptamers and aptamer nanocomplexes, is beginning to fulfill its potential in both clinical trials and preclinical studies. Especially in oncology, aptamer nanomedicine may perform better than conventional or antibody-based chemotherapeutics due to specificity compared to the former and stability compared to the latter. Many proof-of-concept studies on applying aptamers to drug delivery, gene therapy, and cancer imaging have shown promising efficacy and impressive safety in vivo toward translation. Yet, there remains ample room for improvement and critical barriers to be addressed. In this review, we will first introduce the recent progress in clinical trials of aptamer nanomedicine, followed by a discussion of the barriers at the design and in vivo application stages. We will then highlight recent advances and engineering strategies proposed to tackle these barriers. Aptamer cancer nanomedicine has the potential to address one of the most important healthcare issues of the society.

  19. Generation of aptamer for biosensing applications

    Science.gov (United States)

    Gopinath, Subash C. B.; Hashim, U.; Arshad, M. K. Md.; Ruslinda, A. R.

    2016-07-01

    Systematic evolution of ligands by exponential enrichment (SELEX), an in vitro strategy which involves generation of aptamer. Aptamer is an artificial antibody, behave very similar to antibody and several instances reported to be better than antibodies. In this study, an attempt has been made to generate aptamer against factor IX, a potential candidate involve in human blood coagulation cascade. Totally, 10 selection cycles have been performed and molecules from 10th cycle have shown higher binding affinity with factor IX as 56 and 68% against the factor IX concentrations of 100 and 200 nM, respectively. With these higher binding affinities, it is clear that these molecules have higher potential for sensing applications.

  20. Aptamer Based Microsphere Biosensor for Thrombin Detection

    Directory of Open Access Journals (Sweden)

    Xudong Fan

    2006-08-01

    Full Text Available We have developed an optical microsphere resonator biosensor using aptamer asreceptor for the measurement of the important biomolecule thrombin. The sphere surface ismodified with anti-thrombin aptamer, which has excellent binding affinity and selectivityfor thrombin. Binding of the thrombin at the sphere surface is monitored by the spectralposition of the microsphere’s whispering gallery mode resonances. A detection limit on theorder of 1 NIH Unit/mL is demonstrated. Control experiments with non-aptameroligonucleotide and BSA are also carried out to confirm the specific binding betweenaptamer and thrombin. We expect that this demonstration will lead to the development ofhighly sensitive biomarker sensors based on aptamer with lower cost and higher throughputthan current technology.

  1. Aptamer/target binding-induced triple helix forming for signal-on electrochemical biosensing.

    Science.gov (United States)

    Mao, Yinfei; Liu, Jinquan; He, Dinggen; He, Xiaoxiao; Wang, Kemin; Shi, Hui; Wen, Li

    2015-10-01

    Owing to its diversified structures, high affinity, and specificity for binding a wide range of non-nucleic acid targets, aptamer is a useful molecular recognition tool for the design of various biosensors. Herein, we report a new signal-on electrochemical biosensing platform which is based on an aptamer/target binding-induced strand displacement and triple-helix forming. The biosensing platform is composed of a signal transduction probe (STP) modified with a methylene blue (MB) and a sulfhydryl group, a triplex-forming oligonucleotides probe (TFO) and a target specific aptamer probe (Apt). Through hybridization with the TFO probe and the Apt probe, the self-assembled STP on Au electrode via Au-S bonding keeps its rigid structure. The MB on the STP is distal to the Au electrode surface. It is eT off state. Target binding releases the Apt probe and liberates the end of the MB tagged STP to fold back and form a triplex-helix structure with TFO (STP/TFO/STP), allowing MB to approach the Au electrode surface and generating measurable electrochemical signals (eT ON). As test for the feasibility and universality of this signal-on electrochemical biosensing platform, two aptamers which bind to adenosine triphosphate (ATP) and human α-thrombin (Tmb), respectively, are selected as models. The detection limit of ATP was 7.2 nM, whereas the detection limit of Tmb was 0.86 nM.

  2. Improving protein structural class prediction using novel combined sequence information and predicted secondary structural features.

    Science.gov (United States)

    Dai, Qi; Wu, Li; Li, Lihua

    2011-12-01

    Protein structural class prediction solely from protein sequences is a challenging problem in bioinformatics. Numerous efficient methods have been proposed for protein structural class prediction, but challenges remain. Using novel combined sequence information coupled with predicted secondary structural features (PSSF), we proposed a novel scheme to improve prediction of protein structural classes. Given an amino acid sequence, we first transformed it into a reduced amino acid sequence and calculated its word frequencies and word position features to combine novel sequence information. Then we added the PSSF to the combine sequence information to predict protein structural classes. The proposed method was tested on four benchmark datasets in low homology and achieved the overall prediction accuracies of 83.1%, 87.0%, 94.5%, and 85.2%, respectively. The comparison with existing methods demonstrates that the overall improvements range from 2.3% to 27.5%, which indicates that the proposed method is more efficient, especially for low-homology amino acid sequences.

  3. Spectroscopic and Electrochemical Detection of Thrombin/5'-SH or 3'-SH Aptamer Immobilized on (porous) Gold Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Park, Buem Jin; Sa, Young Seung; Kim, Yong Hwan; Kim, Young Hun [Kwangwoon University, Seoul (Korea, Republic of)

    2012-01-15

    Thrombin is a serine protease that catalyzes the conversion of soluble fibrinogen to insoluble fibrin, and thus induces physiological and pathological blood coagulation. Therefore, it is important to detect thrombin in blood serum for purposes of diagnosis. To achieve this goal, it has been suggested that a 15-mer aptamer strongly binds with thrombin to form a G-quartet structure of the aptamer. Generally, 5'-end thiol-functionalized aptamer has been used as an anti-thrombin binder. Herein, we evaluate the possibility of utilizing a 3'-SH aptasensor for thrombin detection using SPR spectroscopy, and compare the enhancement of the electrochemical signal of the thrombin-aptamer bound on a porous gold substrate. Although the two aptamers have similar configurations, in SPR analysis, the 3'-SH aptamer was a effective aptasensor as well as 5'-SH aptamer. Results from electrochemical analysis showed that the porous gold substrate acted as a good substrate for an aptasensor and demonstrated 5-fold enhancement of current change, as compared to gold thin film.

  4. tPA-binding RNA Aptamers

    DEFF Research Database (Denmark)

    Bjerregaard, Nils

    2015-01-01

    nanomolar affinities and efficiently inhibit tPA-LRP-1 binding and LRP-1 mediated cellular endocytosis. Both aptamers minimally affected the fibrinolytic properties of tPA despite efficiently inhibiting plasminogen activation stimulated by a soluble fibrin fragment. K18v2 additionally inhibited plasminogen...... equivalent activity at concentrations reduced by approximately two orders of magnitude. The present results suggests beneficial effects of coadministration of K18v2 or K32v2 in the fibrinolytic treatment of iscahemic stroke, and potential applications of the conjugate aptamer in the management of bleeding...

  5. RNA fluorescence with light-up aptamers

    Science.gov (United States)

    Ouellet, Jonathan

    2016-06-01

    Seeing is not only believing; it also includes understanding. Cellular imaging with GFP in live cells has been transformative in many research fields. Modulation of cellular regulation is tightly regulated and innovative imaging technologies contribute to further understand cellular signaling and physiology. New types of genetically encoded biosensors have been developed over the last decade. They are RNA aptamers that bind with their cognate fluorogen ligands and activate their fluorescence. The emergence and the evolution of these RNA aptamers as well as their conversion into a wide spectrum of applications are examined in a global way.

  6. Data structures of genome and protein sequences indexing

    Directory of Open Access Journals (Sweden)

    Adeleh asadi

    2016-03-01

    Full Text Available Data structure is a tool for storage and retrieval of information which is named logic and mathematic way of specific data organization. various sequences of genes and proteins in various creatures increases the amount of data in genome databases, and finding appropriate data structure and indexing are subject for many studies. String data structures are general data structure for genome indexing, and this article would review the many used three types of string data structure, suffix tree, suffix array, and Directed Acyclic Word Graphs. This paper is a review of the literature related to three types of data, including genome databases indexing field, tree, postfix, postfix and graphs spiral array directly introduces the word. Findings of this research show that suffix tree and Directed Acyclic Word Graph (DAWG structures need much space however suffix array need less space. Against the Directed Acyclic Word Graph, suffix array can be stored on Memory Stick. Suffix tree and Directed Acyclic Word Graph are a dynamic structures but as suffix array is a Sorted out structure, it could hardly be changed.

  7. High-Affinity DNA Aptamer Generation Targeting von Willebrand Factor A1-Domain by Genetic Alphabet Expansion for Systematic Evolution of Ligands by Exponential Enrichment Using Two Types of Libraries Composed of Five Different Bases.

    Science.gov (United States)

    Matsunaga, Ken-Ichiro; Kimoto, Michiko; Hirao, Ichiro

    2017-01-11

    The novel evolutionary engineering method ExSELEX (genetic alphabet expansion for systematic evolution of ligands by exponential enrichment) provides high-affinity DNA aptamers that specifically bind to target molecules, by introducing an artificial hydrophobic base analogue as a fifth component into DNA aptamers. Here, we present a newer version of ExSELEX, using a library with completely randomized sequences consisting of five components: four natural bases and one unnatural hydrophobic base, 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). In contrast to the limited number of Ds-containing sequence combinations in our previous library, the increased complexity of the new randomized library could improve the success rates of high-affinity aptamer generation. To this end, we developed a sequencing method for each clone in the enriched library after several rounds of selection. Using the improved library, we generated a Ds-containing DNA aptamer targeting von Willebrand factor A1-domain (vWF) with significantly higher affinity (KD = 75 pM), relative to those generated by the initial version of ExSELEX, as well as that of the known DNA aptamer consisting of only the natural bases. In addition, the Ds-containing DNA aptamer was stabilized by introducing a mini-hairpin DNA resistant to nucleases, without any loss of affinity (KD = 61 pM). This new version is expected to consistently produce high-affinity DNA aptamers.

  8. A Precise Packing Sequence for Self-Assembled Convex Structures

    Science.gov (United States)

    Chen, Ting; Zhang, Zhenli; Glotzer, Sharon

    2007-03-01

    We present molecular simulations of the self-assembly of cone-shaped particles with patchy, attractive interactions[1,2]. Upon cooling from random initial conditions, we find that the cones self assemble into clusters and that clusters comprised of particular numbers of cones have a unique and precisely packed structure that is robust over a range of cone angles. These precise clusters form precise packing sequence that for small sizes is identical to that observed in evaporation-driven assembly of colloidal spheres. This sequence is reproduced and extended in simulations of two simple models of spheres self-assembling from random initial conditions subject to convexity constraints, and contains six of the most common virus capsid structures obtained in vivo including large chiral clusters, and a cluster that may correspond to several non- icosahedral, spherical virus capsid structures obtained in vivo. For prolate spheroidal convexity conditions, we demonstrate the formation of several prolate virus structures from self-assembling hard spheres[3]. [1] Chen T, Zhang ZL, Glotzer SC, PNAS, in press (http://xxx.lanl.gov/pdf/cond-mat/ 0608592) [2] Chen T, Zhang ZL, Glotzer SC, http://xxx.lanl.gov/pdf/cond-mat/0608613 [3] Chen T, Glotzer SC http://xxx.lanl.gov/pdf/q-bio.BM/0608040

  9. Structure and sequence analysis of influenza A virus nucleoprotein

    Institute of Scientific and Technical Information of China (English)

    NG; Andy; Ka-Leung; SHAW; Pang-Chui

    2009-01-01

    Influenza A virus nucleoprotein (NP) forms homo-oligomers and multiple copies of NP wrap around genomic RNA, along with a trimeric polymerase making up ribonucleoprotein (RNP) complex. Sequence comparison of more than 2500 influenza A NP showed that this protein contains 30.1 % of polymorphic residues. NP is composed of a head and a body domain and a tail loop/ linker region. The head domain is more conserved than the body domain, as revealed from the structure-based sequence alignment. NP oligomerization is mediated by the insertion of the non-polymorphic and structurally conserved tail loop of one NP molecule to a groove of another NP. The different form of NP oligomers is due to the flexibility of the polymorphic linkers that join the tail loop to the rest of the protein. The RNA binding property of NP is known to involve the protruding element and the flexible basic loop between the head and body domains, both having high degree of primary sequence conservation. To bind RNA, NP may first capture the RNA by the flexible basic loop and then the RNA is clamped by the protruding element.

  10. Structure and sequence analysis of influenza A virus nucleoprotein

    Institute of Scientific and Technical Information of China (English)

    NG Andy Ka-Leung; WANG Jia-Huai; SHAW Pang-Chui

    2009-01-01

    Influenza A virus nucleoprotein (NP) forms homo-oligomenrs and multiple copies of NP wrap around genomic RNA, along with a trimeric polymerase making up ribonucleoprotein (RNP) complex. Se-quence comparison of more than 2500 influenza A NP showed that this protein contains 30.1% of po-lymorphic residues. NP is composed of a head and a body domain and a tail loop/linker region. The head domain is more conserved than the body domain, as revealed from the structure-based sequence alignment. NP oligomerization is mediated by the insertion of the non-polymorphic and structurally conserved tail loop of one NP molecule to a groove of another NP. The different form of NP oligomers is due to the flexibility of the polymorphic linkers that join the tail loop to the rest of the protein. The RNA binding property of NP is known to involve the protruding element and the flexible basic loop between the head and body domains, both having high degree of primary sequence conservation. To bind RNA, NP may first capture the RNA by the flexible basic loop and then the RNA is clamped by the protruding element.

  11. A saxitoxin-binding aptamer with higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation.

    Science.gov (United States)

    Zheng, X; Hu, B; Gao, S X; Liu, D J; Sun, M J; Jiao, B H; Wang, L H

    2015-07-01

    Saxitoxin (STX), a member of the family of paralytic shellfish poisoning toxins, poses toxicological and ecotoxicological risks. To develop an analytical recognition element for STX, a DNA aptamer (APT(STX1)) was previously discovered via an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by Handy et al. Our study focused on generating an improved aptamer based on APT(STX1) through rational site-directed mutation and truncation. In this study, we generated the aptamer, M-30f, with a 30-fold higher affinity for STX compared with APT(STX1). The Kd value for M-30f was 133 nM, which was calculated by Bio-Layer Interferometry. After optimization, we detected and compared the interaction of STX with aptamers (APT(STX1) or M-30f) through several techniques (ELISA, cell bioassay, and mouse bioassay). Both aptamers' STX-binding ability was demonstrated in all three methods. Moreover, M-30f performs better than its parent sequence with higher suppressive activity against STX. As a molecular recognition element, M-30f has good prospects for practical application.

  12. Intra-accumbens injection of a dopamine aptamer abates MK-801-induced cognitive dysfunction in a model of schizophrenia.

    Directory of Open Access Journals (Sweden)

    Matthew R Holahan

    Full Text Available Systemic administration of the noncompetitive NMDA-receptor antagonist, MK-801, has been proposed to model cognitive deficits similar to those seen in patients with schizophrenia. The present work investigated the ability of a dopamine-binding DNA aptamer to regulate these MK-801-induced cognitive deficits when injected into the nucleus accumbens. Rats were trained to bar press for chocolate pellet rewards then randomly assigned to receive an intra-accumbens injection of a DNA aptamer (200 nM; n = 7, tris buffer (n = 6 or a randomized DNA oligonucleotide (n = 7. Animals were then treated systemically with MK-801 (0.1 mg/kg and tested for their ability to extinguish their bar pressing response. Two control groups were also included that did not receive MK-801. Data revealed that injection of Tris buffer or the random oligonucleotide sequence into the nucleus accumbens prior to treatment with MK-801 did not reduce the MK-801-induced extinction deficit. Animals continued to press at a high rate over the entire course of the extinction session. Injection of the dopamine aptamer reversed this MK-801-induced elevation in lever pressing to levels as seen in rats not treated with MK-801. Tests for activity showed that the aptamer did not impair locomotor activity. Results demonstrate the in vivo utility of DNA aptamers as tools to investigate neurobiological processes in preclinical animal models of mental health disease.

  13. Intra-accumbens injection of a dopamine aptamer abates MK-801-induced cognitive dysfunction in a model of schizophrenia.

    Science.gov (United States)

    Holahan, Matthew R; Madularu, Dan; McConnell, Erin M; Walsh, Ryan; DeRosa, Maria C

    2011-01-01

    Systemic administration of the noncompetitive NMDA-receptor antagonist, MK-801, has been proposed to model cognitive deficits similar to those seen in patients with schizophrenia. The present work investigated the ability of a dopamine-binding DNA aptamer to regulate these MK-801-induced cognitive deficits when injected into the nucleus accumbens. Rats were trained to bar press for chocolate pellet rewards then randomly assigned to receive an intra-accumbens injection of a DNA aptamer (200 nM; n = 7), tris buffer (n = 6) or a randomized DNA oligonucleotide (n = 7). Animals were then treated systemically with MK-801 (0.1 mg/kg) and tested for their ability to extinguish their bar pressing response. Two control groups were also included that did not receive MK-801. Data revealed that injection of Tris buffer or the random oligonucleotide sequence into the nucleus accumbens prior to treatment with MK-801 did not reduce the MK-801-induced extinction deficit. Animals continued to press at a high rate over the entire course of the extinction session. Injection of the dopamine aptamer reversed this MK-801-induced elevation in lever pressing to levels as seen in rats not treated with MK-801. Tests for activity showed that the aptamer did not impair locomotor activity. Results demonstrate the in vivo utility of DNA aptamers as tools to investigate neurobiological processes in preclinical animal models of mental health disease.

  14. Convergent evolution to an aptamer observed in small populations on DNA microarrays

    Science.gov (United States)

    Rowe, W.; Platt, M.; Wedge, D. C.; Day, P. J. R.; Kell, D. B.; Knowles, J. D.

    2010-09-01

    The development of aptamers on custom synthesized DNA microarrays, which has been demonstrated in recent publications, can facilitate detailed analyses of sequence and fitness relationships. Here we use the technique to observe the paths taken through sequence-fitness space by three different evolutionary regimes: asexual reproduction, recombination and model-based evolution. The different evolutionary runs are made on the same array chip in triplicate, each one starting from a small population initialized independently at random. When evolving to a common target protein, glucose-6-phosphate dehydrogenase (G6PD), these nine distinct evolutionary runs are observed to develop aptamers with high affinity and to converge on the same motif not present in any of the starting populations. Regime specific differences in the evolutions, such as speed of convergence, could also be observed.

  15. Structural insight into the sequence dependence of nucleosome positioning.

    Science.gov (United States)

    Wu, Bin; Mohideen, Kareem; Vasudevan, Dileep; Davey, Curt A

    2010-03-14

    Nucleosome positioning displays sequence dependency and contributes to genomic regulation in a site-specific manner. We solved the structures of nucleosome core particle composed of strong positioning TTTAA elements flanking the nucleosome center. The positioning strength of the super flexible TA dinucleotide is consistent with its observed central location within minor groove inward regions, where it can contribute maximally to energetically challenging minor groove bending, kinking and compression. The marked preference for TTTAA and positioning power of the site 1.5 double helix turns from the nucleosome center relates to a unique histone protein motif at this location, which enforces a sustained, extremely narrow minor groove via a hydrophobic "sugar clamp." Our analysis sheds light on the basis of nucleosome positioning and indicates that the histone octamer has evolved not to fully minimize sequence discrimination in DNA binding.

  16. Fast and low-cost structured light pattern sequence projection.

    Science.gov (United States)

    Wissmann, Patrick; Forster, Frank; Schmitt, Robert

    2011-11-21

    We present a high-speed and low-cost approach for structured light pattern sequence projection. Using a fast rotating binary spatial light modulator, our method is potentially capable of projection frequencies in the kHz domain, while enabling pattern rasterization as low as 2 μm pixel size and inherently linear grayscale reproduction quantized at 12 bits/pixel or better. Due to the circular arrangement of the projected fringe patterns, we extend the widely used ray-plane triangulation method to ray-cone triangulation and provide a detailed description of the optical calibration procedure. Using the proposed projection concept in conjunction with the recently published coded phase shift (CPS) pattern sequence, we demonstrate high accuracy 3-D measurement at 200 Hz projection frequency and 20 Hz 3-D reconstruction rate.

  17. Selection and characterization of DNA aptamers for the development of light-up biosensor to detect Cd(II).

    Science.gov (United States)

    Wang, Hongyan; Cheng, Hui; Wang, Jine; Xu, Lijun; Chen, Hongxia; Pei, Renjun

    2016-07-01

    In order to develop a facile, cost-effective and quick-testing light-up biosensor with excellent specificity for cadmium ions (Cd(II)) detection, a modified selection method based on target-induced release of strands was used to isolate aptamers of Cd (II) with high specificity. Circular Dichroism (CD) data confirmed that one of the selected aptamers underwent a distinct conformational change on addition of Cd (II). A biosensor for Cd(II) was developed based on the Cd(II)-induced release of fluorescence-labeled aptamer from complex with a quencher-labeled short complementary sequence. The sensing platform displayed a Cd(II) concentration-dependent increase of fluorescence intensity in the low micromolar range and had an excellent selectivity in the presence of various interfering metal ions. Such biosensor could potentially be used for the detection of Cd(II) in environmental samples.

  18. Structural Approaches to Sequence Evolution Molecules, Networks, Populations

    CERN Document Server

    Bastolla, Ugo; Roman, H. Eduardo; Vendruscolo, Michele

    2007-01-01

    Structural requirements constrain the evolution of biological entities at all levels, from macromolecules to their networks, right up to populations of biological organisms. Classical models of molecular evolution, however, are focused at the level of the symbols - the biological sequence - rather than that of their resulting structure. Now recent advances in understanding the thermodynamics of macromolecules, the topological properties of gene networks, the organization and mutation capabilities of genomes, and the structure of populations make it possible to incorporate these key elements into a broader and deeply interdisciplinary view of molecular evolution. This book gives an account of such a new approach, through clear tutorial contributions by leading scientists specializing in the different fields involved.

  19. Hinge Atlas: relating protein sequence to sites of structural flexibility

    Directory of Open Access Journals (Sweden)

    Yang Julie

    2007-05-01

    Full Text Available Abstract Background Relating features of protein sequences to structural hinges is important for identifying domain boundaries, understanding structure-function relationships, and designing flexibility into proteins. Efforts in this field have been hampered by the lack of a proper dataset for studying characteristics of hinges. Results Using the Molecular Motions Database we have created a Hinge Atlas of manually annotated hinges and a statistical formalism for calculating the enrichment of various types of residues in these hinges. Conclusion We found various correlations between hinges and sequence features. Some of these are expected; for instance, we found that hinges tend to occur on the surface and in coils and turns and to be enriched with small and hydrophilic residues. Others are less obvious and intuitive. In particular, we found that hinges tend to coincide with active sites, but unlike the latter they are not at all conserved in evolution. We evaluate the potential for hinge prediction based on sequence. Motions play an important role in catalysis and protein-ligand interactions. Hinge bending motions comprise the largest class of known motions. Therefore it is important to relate the hinge location to sequence features such as residue type, physicochemical class, secondary structure, solvent exposure, evolutionary conservation, and proximity to active sites. To do this, we first generated the Hinge Atlas, a set of protein motions with the hinge locations manually annotated, and then studied the coincidence of these features with the hinge location. We found that all of the features have bearing on the hinge location. Most interestingly, we found that hinges tend to occur at or near active sites and yet unlike the latter are not conserved. Less surprisingly, we found that hinge residues tend to be small, not hydrophobic or aliphatic, and occur in turns and random coils on the surface. A functional sequence based hinge predictor was

  20. Topological characterization of crystalline ice structures from coordination sequences

    CERN Document Server

    Herrero, Carlos P

    2013-01-01

    Topological properties of crystalline ice structures are studied by considering ring statistics, coordination sequences, and topological density of different ice phases. The coordination sequences (number of sites at topological distance k from a reference site) have been obtained by direct enumeration until at least 40 coordination spheres for different ice polymorphs. This allows us to study the asymptotic behavior of the mean number of sites in the k-th shell, M_k, for high values of k: M_k ~ a k^2, a being a structure-dependent parameter. Small departures from a strict parabolic dependence have been studied by considering first and second differences of the series {M_k} for each structure. The parameter a ranges from 2.00 for ice VI to 4.27 for ice XII, and is used to define a topological density for these solid phases of water. Correlations between such topological density and the actual volume of ice phases are discussed. Ices Ih and Ic are found to depart from the general trend in this correlation due ...

  1. Method for Confirming Cytoplasmic Delivery of RNA Aptamers

    Science.gov (United States)

    Dickey, David D; Dassie, Justin P; Giangrande, Paloma H

    2016-01-01

    RNA aptamers are single-stranded RNA oligos that represent a powerful emerging technology with potential for treating numerous diseases. More recently, cell-targeted RNA aptamers have been developed for delivering RNA interference (RNAi) modulators (siRNAs and miRNAs) to specific diseased cells (e.g., cancer cells or HIV infected cells) in vitro and in vivo. However, despite initial promising reports, the broad application of this aptamer delivery technology awaits the development of methods that can verify and confirm delivery of aptamers to the cytoplasm of target cells where the RNAi machinery resides. We recently developed a functional assay (RIP assay) to confirm cellular uptake and subsequent cytoplasmic release of an RNA aptamer which binds to a cell surface receptor expressed on prostate cancer cells (PSMA). To assess cytoplasmic delivery, the aptamer was chemically conjugated to saporin, a ribosome inactivating protein toxin that is toxic to cells only when delivered to the cytoplasm (where it inhibits the ribosome) by a cell-targeting ligand (e.g., aptamer). Here, we describe the chemistry used to conjugate the aptamer to saporin and discuss a gel-based method to verify conjugation efficiency. We also detail an in vitro functional assay to confirm that the aptamer retains function following conjugation to saporin and describe a cellular assay to measure aptamer-mediated saporin-induced cytotoxicity. PMID:26472453

  2. Avocado sunblotch viroid: primary sequence and proposed secondary structure.

    Science.gov (United States)

    Symons, R H

    1981-01-01

    The sequence of the 247 nucleotide residues of the single strand circular RNA of avocado sunblotch viroid (ASBV) was determined using partial enzymic cleavage methods on overlapping viroid fragments obtained by partial ribonuclease digestion followed by 32p-labelling in vitro at their 5'-ends. ASBV is much smaller than potato spindle tuber viroid (PSTV; 359 residues) and chrysanthemum stunt viroid (CSV; 356 residues). A secondary structure model for ASBV is proposed and contains 67% of its residues base paired. In contrast to the extensive (69%) sequence homology of CSV with PSTV, only 18% of the ASBV sequence is homologous to PSTV and CSV. There are eight potential polypeptide translation products with chain lengths from 4 to 63 amino acid residues coded for by the plus (infectious) strand and four potential translation products (2 to 60 residues) coded for by the minus strand. An improved method is described for the synthesis of gamma-32p-ATP of high specific activity. PMID:7322921

  3. Delivery, Effect on Cell Viability, and Plasticity of Modified Aptamer Constructs.

    Science.gov (United States)

    Gissberg, Olof; Zaghloul, Eman M; Lundin, Karin E; Nguyen, Chi-Hung; Landras-Guetta, Corinne; Wengel, Jesper; Zain, Rula; Smith, C I Edvard

    2016-06-01

    AS1411 is a g-quadruplex-forming aptamer capable of selectively entering cancer cells by nucleolin receptor-mediated uptake. In this study, we investigated the cell internalization properties and plasticity of AS1411 carrying different locked nucleic acid-containing cargo oligonucleotides (ONs) for delivery into A549 and U2OS cells. We found that internalization efficiency is highly governed by ON cargo chemistry and composition since the inherent antitumor properties of AS1411 were lost when attached to a nontoxic ON, noTox. However, a toxic ON, Tox, demonstrated potent cytotoxicity after aptamer-mediated uptake in A549 cells. We also examined the effect of unlocked nucleic acid (UNA) modifications in the loop region of the aptamer, and how the cargo ONs and UNA incorporation affect the secondary structure of AS1411, in the presence or absence of two novel ellipticine derivatives. These findings add new insights to the design and future applications of aptamer-guided delivery of ON cargo to cancer cells.

  4. Specific detection of oxytetracycline using DNA aptamer-immobilized interdigitated array electrode chip

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yeon Seok; Niazi, Javed H. [School of Life Sciences and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-701 (Korea, Republic of); Gu, Man Bock [School of Life Sciences and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-701 (Korea, Republic of)], E-mail: mbgu@korea.ac.kr

    2009-02-23

    An electrochemical sensing system for oxytetracycline (OTC) detection was developed using ssDNA aptamer immobilized on gold interdigitated array (IDA) electrode chip. A highly specific ssDNA aptamer that bind to OTC with high affinity was employed to discriminate other tetracyclines (TCs), such as doxycycline (DOX) and tetracycline (TET). The immobilized thiol-modified aptamer on gold electrode chip served as a biorecognition element for the target molecules and the electrochemical signals generated from interactions between the aptamers and the target molecules was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV). The current decrease due to the interference of bound OTC, DOX or TET was analyzed with the electron flow produced by a redox reaction between ferro- and ferricyanide. The specificity of developed EC-biosensor for OTC was highly distinguishable from the structurally similar antibiotics (DOX and TET). The dynamic range was determined to be 1-100 nM of OTC concentration in semi-logarithmic coordinates.

  5. Data Structures: Sequence Problems, Range Queries, and Fault Tolerance

    DEFF Research Database (Denmark)

    Jørgensen, Allan Grønlund

    for a range of sequence analysis problems that have risen from applications in pattern matching, bioinformatics, and data mining. On a high level, each problem is dened by a function and some constraints and the job at hand is to locate subsequences that score high with this function and are not invalidated...... a certain function on the elements in a given query subsequence. There are many types of functions that has been considered in connection with input from dierent sources. The input could be ip-data sorted by ip-address, real estate prices sorted by zip code, advertising cost sorted by time etc. We consider...... data structures for two classic statistics functions, namely median and mode. Finally, Part III investigates fault tolerant algorithms and data structures. This deals with the trend of avoiding elaborate error checking and correction circuitry that would impose non-negligible costs in terms of hardware...

  6. Small-Molecule Binding Aptamers: Selection Strategies, Characterization, and Applications

    Science.gov (United States)

    Ruscito, Annamaria; DeRosa, Maria

    2016-05-01

    Aptamers are single-stranded, synthetic oligonucleotides that fold into 3-dimensional shapes capable of binding non-covalently with high affinity and specificity to a target molecule. They are generated via an in vitro process known as the Systematic Evolution of Ligands by EXponential enrichment, from which candidates are screened and characterized, and then applied in aptamer-based biosensors for target detection. Aptamers for small molecule targets such as toxins, antibiotics, molecular markers, drugs, and heavy metals will be the focus of this review. Their accurate detection is ultimately needed for the protection and wellbeing of humans and animals. However, issues such as the drastic difference in size of the aptamer and small molecule make it challenging to select, characterize, and apply aptamers for the detection of small molecules. Thus, recent (since 2012) notable advances in small molecule aptamers, which have overcome some of these challenges, are presented here, while defining challenges that still exist are discussed

  7. Development of universal antidotes to control aptamer activity.

    Science.gov (United States)

    Oney, Sabah; Lam, Ruby T S; Bompiani, Kristin M; Blake, Charlene M; Quick, George; Heidel, Jeremy D; Liu, Joanna Yi-Ching; Mack, Brendan C; Davis, Mark E; Leong, Kam W; Sullenger, Bruce A

    2009-10-01

    With an ever increasing number of people taking numerous medications, the need to safely administer drugs and limit unintended side effects has never been greater. Antidote control remains the most direct means to counteract acute side effects of drugs, but, unfortunately, it has been challenging and cost prohibitive to generate antidotes for most therapeutic agents. Here we describe the development of a set of antidote molecules that are capable of counteracting the effects of an entire class of therapeutic agents based upon aptamers. These universal antidotes exploit the fact that, when systemically administered, aptamers are the only free extracellular oligonucleotides found in circulation. We show that protein- and polymer-based molecules that capture oligonucleotides can reverse the activity of several aptamers in vitro and counteract aptamer activity in vivo. The availability of universal antidotes to control the activity of any aptamer suggests that aptamers may be a particularly safe class of therapeutics.

  8. Aptamers in Bordeaux, 24-25 June 2016.

    Science.gov (United States)

    Toulmé, Jean-Jacques; Giangrande, Paloma H; Mayer, Günter; Suess, Beatrix; Ducongé, Frédéric; Sullenger, Bruce; de Franciscis, Vittorio; Darfeuille, Fabien; Peyrin, Eric

    2017-01-20

    The symposium covered the many different aspects of the selection and the characterization of aptamers as well as their application in analytical, diagnostic and therapeutic areas. Natural and artificial riboswitches were discussed. Recent advances for the design of mutated polymerases and of chemically modified nucleic acid bases that provide aptamers with new properties were presented. The power of aptamer platforms for multiplex analysis of biomarkers of major human diseases was described. The potential of aptamers for the treatment of cancer or cardiovascular diseases was also presented. Brief summaries of the lectures presented during the symposium are given in this report. A second edition of "Aptamers in Bordeaux" will take place on September 2017 (http://www.aptamers-in-bordeaux.com/).

  9. Aptamers in Bordeaux, 24–25 June 2016

    Directory of Open Access Journals (Sweden)

    Jean-Jacques Toulmé

    2017-01-01

    Full Text Available The symposium covered the many different aspects of the selection and the characterization of aptamers as well as their application in analytical, diagnostic and therapeutic areas. Natural and artificial riboswitches were discussed. Recent advances for the design of mutated polymerases and of chemically modified nucleic acid bases that provide aptamers with new properties were presented. The power of aptamer platforms for multiplex analysis of biomarkers of major human diseases was described. The potential of aptamers for the treatment of cancer or cardiovascular diseases was also presented. Brief summaries of the lectures presented during the symposium are given in this report. A second edition of “Aptamers in Bordeaux” will take place on September 2017 (http://www.aptamers-in-bordeaux.com/.

  10. A two-step stimulus-response cell-SELEX method to generate a DNA aptamer to recognize inflamed human aortic endothelial cells as a potential in vivo molecular probe for atherosclerosis plaque detection.

    Science.gov (United States)

    Ji, Kaili; Lim, Wee Siang; Li, Sam Fong Yau; Bhakoo, Kishore

    2013-08-01

    Aptamers are single-stranded oligonucleotides that are capable of binding wide classes of targets with high affinity and specificity. Their unique three-dimensional structures present numerous possibilities for recognizing virtually any class of target molecules, making them a promising alternative to antibodies used as molecular probes in biomedical analysis and clinical diagnosis. In recent years, cell-systematic evolution of ligands by exponential enrichment (SELEX) has been used extensively to select aptamers for various cell targets. However, aptamers that have evolved from cell-SELEX to distinguish the "stimulus-response cell" have not previously been reported. Moreover, a number of cumbersome and time-consuming steps involved in conventional cell-SELEX reduce the efficiency and efficacy of the aptamer selection. Here, we report a "two-step" methodology of cell-SELEX that successfully selected DNA aptamers specifically against "inflamed" endothelial cells. This has been termed as stimulus-response cell-SELEX (SRC-SELEX). The SRC-SELEX enables the selection of aptamers to distinguish the cells activated by stimulus of healthy cells or cells isolated from diseased tissue. We report a promising aptamer, N55, selected by SRC-SELEX, which can bind specifically to inflamed endothelial cells both in cell culture and atherosclerotic plaque tissue. This aptamer probe was demonstrated as a potential molecular probe for magnetic resonance imaging to target inflamed endothelial cells and atherosclerotic plaque detection.

  11. Highly-efficient gating of solid-state nanochannels by DNA supersandwich structure containing ATP aptamers: a nanofluidic IMPLICATION logic device.

    Science.gov (United States)

    Jiang, Yanan; Liu, Nannan; Guo, Wei; Xia, Fan; Jiang, Lei

    2012-09-19

    Integrating biological components into artificial devices establishes an interface to understand and imitate the superior functionalities of the living systems. One challenge in developing biohybrid nanosystems mimicking the gating function of the biological ion channels is to enhance the gating efficiency of the man-made systems. Herein, we demonstrate a DNA supersandwich and ATP gated nanofluidic device that exhibits high ON-OFF ratios (up to 10(6)) and a perfect electric seal at its closed state (~GΩ). The ON-OFF ratio is distinctly higher than existing chemically modified nanofluidic gating systems. The gigaohm seal is comparable with that required in ion channel electrophysiological recording and some lipid bilayer-coated nanopore sensors. The gating function is implemented by self-assembling DNA supersandwich structures into solid-state nanochannels (open-to-closed) and their disassembly through ATP-DNA binding interactions (closed-to-open). On the basis of the reversible and all-or-none electrochemical switching properties, we further achieve the IMPLICATION logic operations within the nanofluidic structures. The present biohybrid nanofluidic device translates molecular events into electrical signals and indicates a built-in signal amplification mechanism for future nanofluidic biosensing and modular DNA computing on solid-state substrates.

  12. Aptamers Against Viral Hepatitis: from Rational Design to Practical Application

    Institute of Scientific and Technical Information of China (English)

    Hui FENG; Kang-hong HU

    2008-01-01

    Aptamers are short nucleic acids or peptides that strongly bind to a protein of interest and functionally inhibit a given target protein at the intracellular level. Besides high affinity and specificity, aptamers have several advantages over traditional antibodies. Hence, they have been broadly selected to develop antiviral agents for therapeutic applications against hepatitis B and C viruses (HBV, HCV). This review provides a summary of in vitro selection and characterization of aptamers against viral hepatitis, which is of practical significance in drug discovery.

  13. Cell-targeting aptamers act as intracellular delivery vehicles.

    Science.gov (United States)

    Gopinath, Subash C B; Lakshmipriya, Thangavel; Chen, Yeng; Arshad, M K Md; Kerishnan, Jesinda P; Ruslinda, A R; Al-Douri, Yarub; Voon, C H; Hashim, Uda

    2016-08-01

    Aptamers are single-stranded nucleic acids or peptides identified from a randomized combinatorial library through specific interaction with the target of interest. Targets can be of any size, from small molecules to whole cells, attesting to the versatility of aptamers for binding a wide range of targets. Aptamers show drug properties that are analogous to antibodies, with high specificity and affinity to their target molecules. Aptamers can penetrate disease-causing microbial and mammalian cells. Generated aptamers that target surface biomarkers act as cell-targeting agents and intracellular delivery vehicles. Within this context, the "cell-internalizing aptamers" are widely investigated via the process of cell uptake with selective binding during in vivo systematic evolution of ligands by exponential enrichment (SELEX) or by cell-internalization SELEX, which targets cell surface antigens to be receptors. These internalizing aptamers are highly preferable for the localization and functional analyses of multiple targets. In this overview, we discuss the ways by which internalizing aptamers are generated and their successful applications. Furthermore, theranostic approaches featuring cell-internalized aptamers are discussed with the purpose of analyzing and diagnosing disease-causing pathogens.

  14. Spectroscopic study of the interaction of insulin and its aptamer – sensitive optical detection of insulin

    Energy Technology Data Exchange (ETDEWEB)

    Verdian-Doghaei, A.; Housaindokht, M.R., E-mail: housain@um.ac.ir

    2015-03-15

    The binding of insulin to the insulin binding aptamer (IBA) was investigated with different spectroscopy techniques (UV/vis absorption, fluorescence, resonance light-scattering spectra (RLS), synchronous fluorescence, and three-dimensional fluorescence). The thermodynamic parameters (K{sub b}, ΔH, ΔG, and ΔS) of the insulin–IBA complex were further investigated by temperature-dependent fluorescence measurement over the range of 25–37 °C. The results of synchronous fluorescence, resonance light scattering (RLS) and three-dimensional fluorescence spectra showed that IBA would alter the structure of insulin. Folding of the dual-labeled insulin binding aptamer, FL-IBA, carrying 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) labels at termini of the insulin binding aptamer (IBA), into G-quadruplex leading to the contact quenching occurs as a result of the formation of a nonfluorescent complex between donor and acceptor. Significant fluorescent signal change when FL-IBA was bound to insulin is attributed to a conformational change in FL-IBA from a loose random coil to a compact G-quadruplex. Based on fluorescence quenching of IBA folding, a simple and sensitive insulin aptamer-based biosensor in the range of 2–70 nM was proposed. - Highlights: • The binding parameters were gotten from contact quenching data. • The binding of IBA induced some microenvironment and conformational changes in insulin. • The van der Waals interactions or hydrogen bonds play a major role on this binding. • A simple and sensitive insulin aptamer-based biosensor was proposed.

  15. Structure and sequence of the saimiriine herpesvirus 1 genome.

    Science.gov (United States)

    Tyler, Shaun; Severini, Alberto; Black, Darla; Walker, Matthew; Eberle, R

    2011-02-05

    We report here the complete genome sequence of the squirrel monkey α-herpesvirus saimiriine herpesvirus 1 (HVS1). Unlike the simplexviruses of other primate species, only the unique short region of the HVS1 genome is bounded by inverted repeats. While all Old World simian simplexviruses characterized to date lack the herpes simplex virus RL1 (γ34.5) gene, HVS1 has an RL1 gene. HVS1 lacks several genes that are present in other primate simplexviruses (US8.5, US10-12, UL43/43.5 and UL49A). Although the overall genome structure appears more like that of varicelloviruses, the encoded HVS1 proteins are most closely related to homologous proteins of the primate simplexviruses. Phylogenetic analyses confirm that HVS1 is a simplexvirus. Limited comparison of two HVS1 strains revealed a very low degree of sequence variation more typical of varicelloviruses. HVS1 is thus unique among the primate α-herpesviruses in that its genome has properties of both simplexviruses and varicelloviruses.

  16. Nucleotide sequence of the structural gene for tryptophanase of Escherichia coli K-12.

    OpenAIRE

    Deeley, M C; Yanofsky, C

    1981-01-01

    The tryptophanase structural gene, tnaA, of Escherichia coli K-12 was cloned and sequenced. The size, amino acid composition, and sequence of the protein predicted from the nucleotide sequence agree with protein structure data previously acquired by others for the tryptophanase of E. coli B. Physiological data indicated that the region controlling expression of tnaA was present in the cloned segment. Sequence data suggested that a second structural gene of unknown function was located distal ...

  17. A novel predictor for protein structural class based on integrated information of the secondary structure sequence.

    Science.gov (United States)

    Zhang, Lichao; Zhao, Xiqiang; Kong, Liang; Liu, Shuxia

    2014-08-01

    The structural class has become one of the most important features for characterizing the overall folding type of a protein and played important roles in many aspects of protein research. At present, it is still a challenging problem to accurately predict protein structural class for low-similarity sequences. In this study, an 18-dimensional integrated feature vector is proposed by fusing the information about content and position of the predicted secondary structure elements. The consistently high accuracies of jackknife and 10-fold cross-validation tests on different low-similarity benchmark datasets show that the proposed method is reliable and stable. Comparison of our results with other methods demonstrates that our method is an effective computational tool for protein structural class prediction, especially for low-similarity sequences.

  18. Identification of similar regions of protein structures using integrated sequence and structure analysis tools

    Directory of Open Access Journals (Sweden)

    Heiland Randy

    2006-03-01

    Full Text Available Abstract Background Understanding protein function from its structure is a challenging problem. Sequence based approaches for finding homology have broad use for annotation of both structure and function. 3D structural information of protein domains and their interactions provide a complementary view to structure function relationships to sequence information. We have developed a web site http://www.sblest.org/ and an API of web services that enables users to submit protein structures and identify statistically significant neighbors and the underlying structural environments that make that match using a suite of sequence and structure analysis tools. To do this, we have integrated S-BLEST, PSI-BLAST and HMMer based superfamily predictions to give a unique integrated view to prediction of SCOP superfamilies, EC number, and GO term, as well as identification of the protein structural environments that are associated with that prediction. Additionally, we have extended UCSF Chimera and PyMOL to support our web services, so that users can characterize their own proteins of interest. Results Users are able to submit their own queries or use a structure already in the PDB. Currently the databases that a user can query include the popular structural datasets ASTRAL 40 v1.69, ASTRAL 95 v1.69, CLUSTER50, CLUSTER70 and CLUSTER90 and PDBSELECT25. The results can be downloaded directly from the site and include function prediction, analysis of the most conserved environments and automated annotation of query proteins. These results reflect both the hits found with PSI-BLAST, HMMer and with S-BLEST. We have evaluated how well annotation transfer can be performed on SCOP ID's, Gene Ontology (GO ID's and EC Numbers. The method is very efficient and totally automated, generally taking around fifteen minutes for a 400 residue protein. Conclusion With structural genomics initiatives determining structures with little, if any, functional characterization

  19. SCPRED: Accurate prediction of protein structural class for sequences of twilight-zone similarity with predicting sequences

    Directory of Open Access Journals (Sweden)

    Chen Ke

    2008-05-01

    Full Text Available Abstract Background Protein structure prediction methods provide accurate results when a homologous protein is predicted, while poorer predictions are obtained in the absence of homologous templates. However, some protein chains that share twilight-zone pairwise identity can form similar folds and thus determining structural similarity without the sequence similarity would be desirable for the structure prediction. The folding type of a protein or its domain is defined as the structural class. Current structural class prediction methods that predict the four structural classes defined in SCOP provide up to 63% accuracy for the datasets in which sequence identity of any pair of sequences belongs to the twilight-zone. We propose SCPRED method that improves prediction accuracy for sequences that share twilight-zone pairwise similarity with sequences used for the prediction. Results SCPRED uses a support vector machine classifier that takes several custom-designed features as its input to predict the structural classes. Based on extensive design that considers over 2300 index-, composition- and physicochemical properties-based features along with features based on the predicted secondary structure and content, the classifier's input includes 8 features based on information extracted from the secondary structure predicted with PSI-PRED and one feature computed from the sequence. Tests performed with datasets of 1673 protein chains, in which any pair of sequences shares twilight-zone similarity, show that SCPRED obtains 80.3% accuracy when predicting the four SCOP-defined structural classes, which is superior when compared with over a dozen recent competing methods that are based on support vector machine, logistic regression, and ensemble of classifiers predictors. Conclusion The SCPRED can accurately find similar structures for sequences that share low identity with sequence used for the prediction. The high predictive accuracy achieved by SCPRED is

  20. An Improved Protein Structural Prediction Method by Incorporating Both Sequence and Structure Information.

    Science.gov (United States)

    Wei, Leyi; Liao, Minghong; Gao, Xing; Zou, Quan

    2014-09-15

    Protein structural classes information is beneficial for secondary and tertiary structure prediction, protein folds prediction, and protein function analysis. Thus, predicting protein structural classes is of vital importance. In recent years, several computational methods have been developed for low-sequence-similarity (25% - 40%) protein structural classes prediction. However, the reported prediction accuracies are actually not satisfactory. Aiming to further improve the prediction accuracies, we propose three different feature extraction methods and construct a comprehensive feature set that captures both sequence and structure information. By applying a random forest (RF) classifier to the feature set, we further develop a novel method for structural classes prediction. We test the proposed method on three benchmark datasets (25PDB, 640, and 1189) with low sequence similarity, and obtain the overall prediction accuracies of 93.5%, 92.6%, and 93.4%, respectively. Compared with six competing methods, the accuracies we achieved are 3.4%, 6.2%, and 8.7% higher than that achieved by the best-performing methods, showing the superiority of our method. Moreover, due to the limitation of the size of the three benchmark datasets, we further test the proposed method on three updated large-scale datasets with different sequence similarity (40%, 30%, and 25%). The proposed method achieves above 90% accuracies for all the three datasets, consistency with the accuracies on the above three benchmark datasets. Experimental results suggest our method as an effective and promising tool for structural classes prediction. Currently, a webserver that implements the proposed method is available on http://121.192.180.204:8080/RF_PSCP/Index.html.

  1. Aptamer Stainings for Super-resolution Microscopy.

    Science.gov (United States)

    de Castro, Maria Angela Gomes; Rammner, Burkhard; Opazo, Felipe

    2016-01-01

    Fluorescence microscopy is an invaluable tool to visualize molecules in their biological context with ease and flexibility. However, studies using conventional light microscopy have been limited to the resolution that light diffraction allows (i.e., ~200 nm). This limitation has been recently circumvented by several types of advanced fluorescence microscopy techniques, which have achieved resolutions of up to ~10 nm. The resulting enhanced imaging precision has helped to find important cellular details that were not visible using diffraction-limited instruments. However, it has also revealed that conventional stainings using large affinity tags, such as antibodies, are not accurate enough for these imaging techniques. Since aptamers are substantially smaller than antibodies, they could provide a real advantage in super-resolution imaging. Here we compare the live staining of transferrin receptors (TfnR) obtained with different fluorescently labeled affinity probes: aptamers, specific monoclonal antibodies, or the natural receptor ligand transferrin. We observed negligible differences between these staining strategies when imaging is performed with conventional light microscopy (i.e., laser scanning confocal microscopy). However, a clear superiority of the aptamer tag over antibodies became apparent in super-resolved images obtained with stimulated emission depletion (STED) microscopy.

  2. An Analysis of the Syllabic Structure of English Mute in the Light of Sonority Sequencing Principle

    Institute of Scientific and Technical Information of China (English)

    郑双

    2015-01-01

    Syllable is the hierarchic natural feature of a language,and there are rules of the phonological sequences of the syllabic structure.Based on the Sonority Sequencing Principle (SSP),this paper will try to discuss and analyze phonological sequences of the syllabic structure of English mutes.

  3. An Analysis of the Syllabic Structure of English Mute in the Light of Sonority Sequencing Principle

    Institute of Scientific and Technical Information of China (English)

    郑双

    2015-01-01

    Syllable is the hierarchic natural feature of a language,and there are rules of the phonological sequences of the syllabic structure.Based on the Sonority Sequencing Principle(SSP),this paper will try to discuss and analyze phonological sequences of the syllabic structure of English mutes.

  4. Modular Assembly of Cell-targeting Devices Based on an Uncommon G-quadruplex Aptamer

    DEFF Research Database (Denmark)

    Opazo, Felipe; Eiden, Laura; Hansen, Line;

    2015-01-01

    cells. We further optimized this aptamer to a highly versatile and stable minimized version. The minimized aptamer can be easily equipped with different functionalities like quantum dots, organic dyes, or even a second different aptamer domain yielding a bi-paratopic aptamer. Although the target...

  5. An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates.

    Science.gov (United States)

    Padlan, Camille S; Malashkevich, Vladimir N; Almo, Steve C; Levy, Matthew; Brenowitz, Michael; Girvin, Mark E

    2014-04-01

    RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid-protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Our 2.0-Å crystal structure of the aptamer-protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein-nucleic acid interface; (1) only 410 Å(2) of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (∼18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na(+) stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE-lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform.

  6. All-atom molecular dynamics insights on preQ1 riboswitch aptamer

    Science.gov (United States)

    Gong, Zhou; Zhao, Yunjie; Chen, Changjun; Xiao, Yi

    2012-02-01

    Recently, a series of experiments have focused on two types of preQ1 riboswitch with known smallest aptamer. One of them is from Bacillus subtilis, which have been discussed before. The other one comes from T. tengcongensis, and Jenkins et al recently release its crystal structure in both ligand-bound and free state. These two types of riboswitch aptamer have similar structures but totally different functions. Consequently, contrast studies of these two preQ1 riboswitches will help us to understand the regulation function of riboswitch better. Here, we study the dynamical properties of two types of preQ1 riboswitches using molecular dynamics simulation. We find that the unfolding pathway of the two preQ1 aptamer domains in bound state are both hierarchical and have an intermediate state. We believe that such conformation would be a good candidate structure for ligand binding. On the other hand, in the absent of ligand, the preQ1 riboswitch from Bacillus subtilis can only form the stable state with P1-L3 triplex, while the preQ1 riboswitch from T. tengcongensis can form the conformation with pseudoknot shape. We suggest that such intermediate structures may perform regulation functions in the absent of ligand.

  7. Aptamers as Both Drugs and Drug-Carriers

    Directory of Open Access Journals (Sweden)

    Md. Ashrafuzzaman

    2014-01-01

    Full Text Available Aptamers are short nucleic acid oligos. They may serve as both drugs and drug-carriers. Their use as diagnostic tools is also evident. They can be generated using various experimental, theoretical, and computational techniques. The systematic evolution of ligands by exponential enrichment which uses iterative screening of nucleic acid libraries is a popular experimental technique. Theory inspired methodology entropy-based seed-and-grow strategy that designs aptamer templates to bind specifically to targets is another one. Aptamers are predicted to be highly useful in producing general drugs and theranostic drugs occasionally for certain diseases like cancer, Alzheimer’s disease, and so on. They bind to various targets like lipids, nucleic acids, proteins, small organic compounds, and even entire organisms. Aptamers may also serve as drug-carriers or nanoparticles helping drugs to get released in specific target regions. Due to better target specific physical binding properties aptamers cause less off-target toxicity effects. Therefore, search for aptamer based drugs, drug-carriers, and even diagnostic tools is expanding fast. The biophysical properties in relation to the target specific binding phenomena of aptamers, energetics behind the aptamer transport of drugs, and the consequent biological implications will be discussed. This review will open up avenues leading to novel drug discovery and drug delivery.

  8. Application of aptamers in diagnostics, drug-delivery and imaging

    Indian Academy of Sciences (India)

    CHETAN CHANDOLA; SHEETAL KALME; MARCO G CASTELEIJN; ARTO URTTI; MUNIASAMY NEERATHILINGAM

    2016-09-01

    Aptamers are small, single-stranded oligonucleotides (DNA or RNA) that bind to their target with high specificity andaffinity. Although aptamers are analogous to antibodies for a wide range of target recognition and variety ofapplications, they have significant advantages over antibodies. Since aptamers have recently emerged as a class ofbiomolecules with an application in a wide array of fields, we need to summarize the latest developments herein. Inthis review we will discuss about the latest developments in using aptamers in diagnostics, drug delivery and imaging.We begin with diagnostics, discussing the application of aptamers for the detection of infective agents itself, antigens/toxins (bacteria), biomarkers (cancer), or a combination. The ease of conjugation and labelling of aptamers makesthem a potential tool for diagnostics. Also, due to the reduced off-target effects of aptamers, their use as a potentialdrug delivery tool is emerging rapidly. Hence, we discuss their use in targeted delivery in conjugation with siRNAs,nanoparticles, liposomes, drugs and antibodies. Finally, we discuss about the conjugation strategies applicable forRNA and DNA aptamers for imaging. Their stability and self-assembly after heating makes them superior overprotein-based binding molecules in terms of labelling and conjugation strategies.

  9. A DNA Structure-Based Bionic Wavelet Transform and Its Application to DNA Sequence Analysis

    Directory of Open Access Journals (Sweden)

    Fei Chen

    2003-01-01

    Full Text Available DNA sequence analysis is of great significance for increasing our understanding of genomic functions. An important task facing us is the exploration of hidden structural information stored in the DNA sequence. This paper introduces a DNA structure-based adaptive wavelet transform (WT – the bionic wavelet transform (BWT – for DNA sequence analysis. The symbolic DNA sequence can be separated into four channels of indicator sequences. An adaptive symbol-to-number mapping, determined from the structural feature of the DNA sequence, was introduced into WT. It can adjust the weight value of each channel to maximise the useful energy distribution of the whole BWT output. The performance of the proposed BWT was examined by analysing synthetic and real DNA sequences. Results show that BWT performs better than traditional WT in presenting greater energy distribution. This new BWT method should be useful for the detection of the latent structural features in future DNA sequence analysis.

  10. Mesoporous materials modified by aptamers and hydrophobic groups assist ultra-sensitive insulin detection in serum.

    Science.gov (United States)

    Lei, Chang; Xu, Chun; Noonan, Owen; Meka, Anand Kumar; Zhang, Long; Nouwens, Amanda; Yu, Chengzhong

    2015-09-14

    A novel mesoporous material modified with both insulin-binding-aptamers and hydrophobic methyl groups is synthesized. With rationally designed pore structures and surface chemistry, this material is applied in sample pre-treatment for ELISA, and enables the quantification (0.25-5 pg ml(-1)) of insulin in serum, 30-fold enhancement of the limit-of-detection compared to the commercial ELISA kit.

  11. Optimization of SELEX: comparison of different methods for monitoring the progress of in vitro selection of aptamers.

    Science.gov (United States)

    Mencin, Nina; Šmuc, Tina; Vraničar, Marko; Mavri, Jan; Hren, Matjaž; Galeša, Katja; Krkoč, Peter; Ulrich, Henning; Šolar, Borut

    2014-03-01

    Oligonucleotide aptamers are selected from libraries typically comprising up to 10(15) different sequences by an iterative process of binding, separation, amplification and purification, called SELEX. During this process, the diversity of the oligonucleotide pool decreases until, presumably, only sequences with highest binding affinities towards chosen targets remain. This selection technique is time-consuming, labor-intensive and expensive. Though well posed in principles, the SELEX procedure is noise sensitive, due to amplification of unspecific-binding sequences, and it is not surprising that aptamer selection is often not successful in practice. In view of that, a follow-up of the progress of selection during its course with simple yet reliable methods is necessary. In this paper, we describe five independent assays to estimate the sequence complexity of SELEX pools including qualitative restriction fragment length polymorphism analysis, melting curve analysis, quantitative fluorescence intensity measurements of bound ssDNA, real time PCR quantification and pool dissociation constant analysis during the progress of aptamer selection against streptavidin. Properties and features of each method are discussed and compared. Pool dissociation constant analysis and sequencing serve as reference methods.

  12. Calcineurin A versus NS5A-TP2/HD domain containing 2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers.

    Science.gov (United States)

    Dibenedetto, Silvia; Cluet, David; Stebe, Pierre-Nicolas; Baumle, Véronique; Léault, Jérémie; Terreux, Raphaël; Bickle, Marc; Chassey, Benoit D E; Mikaelian, Ivan; Colas, Pierre; Spichty, Martin; Zoli, Michele; Rudkin, Brian B

    2013-07-01

    We previously identified a peptide aptamer (named R5G42) via functional selection for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using R5G42 as "bait," allowed the identification of two binding proteins with very different functions: calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized for its role in the immune response, and NS5A-TP2/HD domain containing 2, a much less studied protein induced subsequent to hepatitis C virus non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective in the present study was to dissect the dual target specificity of R5G42 in order to have tools with which to better characterize the actions of the peptide aptamers toward their individual targets. This was achieved through the selection of random mutants of the variable loop, derived from R5G42, evaluating their specificity toward CnA and NS5A-TP2 and analyzing their sequence. An interdisciplinary approach involving biomolecular computer simulations with integration of the sequence data and yeast two-hybrid binding phenotypes of these mutants yielded two structurally distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA- versus NS5A-TP2-specific peptide aptamers indicated that although both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate the nuclear translocation of nuclear factor of activated T cells, indicative of the activation of endogenous CnA. By dissecting the target specificity of R5G42, we have generated novel tools with which to study each target individually. Apta-C8 is capable of directly activating CnA independent of binding to NS5A-TP2 and will be an important tool in studying the role of CnA activation in the regulation of different signaling pathways, whereas Apta-E1 will allow dissection of the function of NS5A

  13. Aptamer-assembled nanomaterials for fluorescent sensing and imaging

    Science.gov (United States)

    Lu, Danqing; He, Lei; Zhang, Ge; Lv, Aiping; Wang, Ruowen; Zhang, Xiaobing; Tan, Weihong

    2017-01-01

    Aptamers, which are selected in vitro by a technology known as the systematic evolution of ligands by exponential enrichment (SELEX), represent a crucial recognition element in molecular sensing. With advantages such as good biocompatibility, facile functionalization, and special optical and physical properties, various nanomaterials can protect aptamers from enzymatic degradation and nonspecific binding in living systems and thus provide a preeminent platform for biochemical applications. Coupling aptamers with various nanomaterials offers many opportunities for developing highly sensitive and selective sensing systems. Here, we focus on the recent applications of aptamer-assembled nanomaterials in fluorescent sensing and imaging. Different types of nanomaterials are examined along with their advantages and disadvantages. Finally, we look toward the future of aptamer-assembled nanomaterials.

  14. Aptamers Against Immunologic Targets: Diagnostic and Therapeutic Prospects.

    Science.gov (United States)

    Vorobyeva, Mariya; Timoshenko, Valentina; Vorobjev, Pavel; Venyaminova, Alya

    2016-02-01

    The concept of in vitro selection of nucleic acid aptamers emerged 25 years ago, and since then tremendous progress has been achieved in the development of different aptamers and their applications for various bioanalytical and therapeutic purposes. Among other protein targets of aptamers, immune system proteins are of particular interest both as diagnostic markers and therapeutic targets. The present review summarizes up-to-date articles concerning the selection and design of DNA and RNA aptamers against immunologic targets such as antibodies, cytokines, and T-cell and B-cell receptors. We also discuss the prospects of employing aptamers as recognizing modules of diagnostic aptasensors, potential therapeutic candidates for the treatment of autoimmune diseases and cancer, and specific tools for functional studies of immune system proteins.

  15. Development of anti β glucan aptamers for use as radiopharmaceutical in the identification of fungal Infections

    Energy Technology Data Exchange (ETDEWEB)

    Lacerda, Camila Maria de Sousa; Reis, Mariana Flister; Correa, Cristiane Rodrigues; Andrade, Antero S.R., E-mail: cmsl@cdtn.br, E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEM-MG), Belo Horizonte, MG (Brazil)

    2013-07-01

    Invasive fungal infections caused by Candida albicans, are recognized as a major cause of morbidity and mortality in immuno compromised individuals. Patients may not show obvious clinical signs or symptoms, making it difficult to detect its origin or new focus that developed through hematogenous spread. Nuclear medicine could contribute to an early diagnosis of fungal infections, since specific markers are available. The aim of this study was to develop, through SELEX technique (Systematic Evolution of Ligands by Exponential Enrichment), aptamers for beta glucan for subsequent labeling with {sup 99}mTc and evaluation of this radiopharmaceutical in the diagnosis of invasive fungal infections, scintigraphy. To obtain aptamers were performed 15 cycles of SELEX technique, using centrifugation as separation method of oligonuclotideos linked to the beta-glucan is not connected. The DNA bands were observed in all 15 cycles. The oligonucleotides obtained after cycles were cloned using the standard protocol kit-Topo TA vector (Invitrogen), and subjected to sequencing Megabase. Three aptamers for yeast cells were selected for this study. Further, other studies should be performed to assess the specificity and affinity thereof for later use in the diagnosis of fungal infections. (author)

  16. Characterization of an RNA aptamer against HPV-16 L1 virus-like particles.

    Science.gov (United States)

    Leija-Montoya, Ana Gabriela; Benítez-Hess, María Luisa; Toscano-Garibay, Julia Dolores; Alvarez-Salas, Luis Marat

    2014-10-01

    The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3'end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection.

  17. In vitro selection of DNA aptamers targeting β-lactoglobulin and their integration in graphene-based biosensor for the detection of milk allergen.

    Science.gov (United States)

    Eissa, Shimaa; Zourob, Mohammed

    2017-05-15

    Food allergy has increased rapidly in recent years affecting millions of people worldwide. With the increased consumption of packed food nowadays, a sensitive, accurate and rapid screening method for potential food allergens has become an urgent need in order to protect the sensitive consumers from life-threatening reactions. The current detection methods for food allergens are mostly based on immunoassays which are costly and times-consuming. In this work, we developed an aptamer/graphene-based electrochemical biosensor for on-step, sensitive and low cost detection of β-lactoglobulin (β-LG) milk protein, one of the most common food allergens specially in infants. The selection of DNA aptamers against the two β-LG variants A and B was successfully realised using systemic evolution of ligands by exponential enrichment (SELEX) method. Among the selected aptamers, BLG14 aptamer sequence has shown high affinity and specificity to both β-LG A and B with dissociation constants (Kds) of 82 and 80nM, respectively as calculated using fluorescence binding assays. The aptamer was then integrated in a voltammetric biosensor utilizing graphene-modified screen printed carbon electrodes. The binding is monitored by following the change in the square wave voltammetry (SWV) reduction peak signal of ferrocyanide/ferricyanide redox couple due to the removal of the negatively charged aptamers from the surface upon protein binding. This one-step "signal on" biosensor takes 20min for the sensitive and selective detection of β-LG without using any labelling or sophisticated designs. The method was also tested in spiked food sample extract achieving good recovery percentage. The integration of the novel aptamer in the graphene biosensor allows a promising way for cost-effective, rapid and sensitive on-site detection of milk allergen in food stuff.

  18. Selection and Application of ssDNA Aptamers against Clenbuterol Hydrochloride Based on ssDNA Library Immobilized SELEX.

    Science.gov (United States)

    Duan, Nuo; Gong, Wenhui; Wu, Shijia; Wang, Zhouping

    2017-03-01

    Clenbuterol hydrochloride (CLB) is often abused as additive feed for livestock to decrease adipose tissue deposition and to increase growth rate. It raises a potential risk to human health through the consumption of animal product. In this study, aptamers with higher affinity and specificity were screened through 16 selection rounds based on the ssDNA library immobilized systematic evolution of ligands by exponential enrichment (SELEX) technique. After cloning and sequencing, five aptamer candidates were picked out for affinity and specificity assays based on a graphene oxide (GO) adsorption method. The results showed that the aptamer CLB-2 binds specifically against CLB with a dissociation constant, Kd, value of 76.61 ± 12.70 nM. In addition, an aptamer-based fluorescence bioassay was established for CLB analysis. The correlation between the CLB concentration and fluorescent signal was found to be linear within the range of 0.10 to 50 ng/mL with a limit of detection of 0.07 ng/mL. It has been further applied for the determination of CLB in pork samples, showing its great potential for sensitive analysis in food safety control.

  19. PROMALS3D: multiple protein sequence alignment enhanced with evolutionary and three-dimensional structural information.

    Science.gov (United States)

    Pei, Jimin; Grishin, Nick V

    2014-01-01

    Multiple sequence alignment (MSA) is an essential tool with many applications in bioinformatics and computational biology. Accurate MSA construction for divergent proteins remains a difficult computational task. The constantly increasing protein sequences and structures in public databases could be used to improve alignment quality. PROMALS3D is a tool for protein MSA construction enhanced with additional evolutionary and structural information from database searches. PROMALS3D automatically identifies homologs from sequence and structure databases for input proteins, derives structure-based constraints from alignments of three-dimensional structures, and combines them with sequence-based constraints of profile-profile alignments in a consistency-based framework to construct high-quality multiple sequence alignments. PROMALS3D output is a consensus alignment enriched with sequence and structural information about input proteins and their homologs. PROMALS3D Web server and package are available at http://prodata.swmed.edu/PROMALS3D.

  20. Training set reduction methods for protein secondary structure prediction in single-sequence condition

    OpenAIRE

    2007-01-01

    Orphan proteins are characterized by the lack of significant sequence similarity to database proteins. To infer the functional properties of the orphans, more elaborate techniques that utilize structural information are required. In this regard, the protein structure prediction gains considerable importance. Secondary structure prediction algorithms designed for orphan proteins (also known as single-sequence algorithms) cannot utilize multiple alignments or alignment prof...

  1. Graphene-Assisted Label-Free Homogeneous Electrochemical Biosensing Strategy based on Aptamer-Switched Bidirectional DNA Polymerization.

    Science.gov (United States)

    Wang, Wenxiao; Ge, Lei; Sun, Ximei; Hou, Ting; Li, Feng

    2015-12-30

    In this contribution, taking the discrimination ability of graphene over single-stranded (ss) DNA/double-stranded (ds) DNA in combination with the electrochemical impedance transducer, we developed a novel label-free homogeneous electrochemical biosensor using graphene-modified glassy carbon electrode (GCE) as the sensing platform. To convert the specific aptamer-target recognition into ultrasensitive electrochemical signal output, a novel aptamer-switched bidirectional DNA polymerization (BDP) strategy, capable of both target recycling and exponential signal amplification, was compatibly developed in this study. In this strategy, all the designed DNA structures could be adsorbed on the graphene/GCE and, thus, serve as the electrochemical impedance signal reporter, while the target acts as a trigger of this BDP reaction, in which these designed DNA structures are bound together and, then, converted to long dsDNA duplex. The distinct difference in electrochemical impedance spectroscopy between the designed structures and generated long dsDNA duplex on the graphene/GCE allows label-free and homogeneous detection of target down to femto-gram level. The target can be displaced from aptamer through the polymerization to initiate the next recognition-polymerization cycle. Herein, the design and signaling principle of aptamer-switched BDP amplification system were elucidated, and the working conditions were optimized. This method not only provides a universal platform for electrochemical biosensing but also shows great potential in biological process researches and clinic diagnostics.

  2. High-Throughput Sequencing Based Methods of RNA Structure Investigation

    DEFF Research Database (Denmark)

    Kielpinski, Lukasz Jan

    describe several computational methods. One that alleviates PCR bias by estimating number of unique molecules existing before the amplification, and two methods for data normalization: one applicable when the paired end sequencing is performed, and the other that works with the single read sequencing...

  3. Intracellular Monitoring of AS1411 Aptamer by Time-Resolved Microspectrofluorimetry and Fluorescence Imaging.

    Science.gov (United States)

    Kočišová, Eva; Praus, Petr; Bok, Jiří; Bonneau, Stéphanie; Sureau, Franck

    2015-09-01

    Time-resolved microspectrofluorimetry and fluorescence microscopy imaging-two complementary fluorescence techniques-provide important information about the intracellular distribution, level of uptake and binding/interactions inside living cell of the labeled molecule of interest. They were employed to monitor the "fate" of AS1411 aptamer labeled by ATTO 425 in human living cells. Confocal microspectrofluorimeter adapted for time-resolved intracellular fluorescence measurements by using a phase-modulation principle with homodyne data acquisition was employed to obtain emission spectra and to determine fluorescence lifetimes in U-87 MG tumor brain cells and Hs68 non-tumor foreskin cells. Acquired spectra from both the intracellular space and the reference solutions were treated to observe the aptamer localization and its interaction with biological structures inside the living cell. The emission spectra and the maximum emission wavelengths coming from the cells are practically identical, however significant lifetime lengthening was observed for tumor cell line in comparison to non-tumor one.

  4. Detection of Interferon gamma using graphene and aptamer based FET-like electrochemical biosensor.

    Science.gov (United States)

    Farid, Sidra; Meshik, Xenia; Choi, Min; Mukherjee, Souvik; Lan, Yi; Parikh, Devanshi; Poduri, Shripriya; Baterdene, Undarmaa; Huang, Ching-En; Wang, Yung Yu; Burke, Peter; Dutta, Mitra; Stroscio, Michael A

    2015-09-15

    One of the primary goals in the scientific community is the specific detection of proteins for the medical diagnostics and biomedical applications. Interferon-gamma (IFN-γ) is associated with the tuberculosis susceptibility, which is one of the major health problems globally. We have therefore developed a DNA aptamer-based electrochemical biosensor that is used for the detection of IFN-γ with high selectivity and sensitivity. A graphene monolayer-based FET-like structure is incorporated on a PDMS substrate with the IFN-γ aptamer attached to graphene. Addition of target molecule induces a change in the charge distribution in the electrolyte, resulting in increase in electron transfer efficiency that was actively sensed by monitoring the change in current from the device. Change in current appears to be highly sensitive to the IFN-γ concentrations ranging from nanomolar (nM) to micromolar (μM) range. The detection limit of our IFN-γ electrochemical biosensor is found to be 83 pM. Immobilization of aptamer on graphene surface is verified using unique structural approach by Atomic Force Microscopy. Such simple and sensitive electrochemical biosensor has potential applications in infectious disease monitoring, immunology and cancer research in the future.

  5. Large cryptic internal sequence repeats in protein structures from Homo sapiens

    Indian Academy of Sciences (India)

    R Sarani; N A Udayaprakash; R Subashini; P Mridula; T Yamane; K Sekar

    2009-03-01

    Amino acid sequences are known to constantly mutate and diverge unless there is a limiting condition that makes such a change deleterious. However, closer examination of the sequence and structure reveals that a few large, cryptic repeats are nevertheless sequentially conserved. This leads to the question of why only certain repeats are conserved at the sequence level. It would be interesting to find out if these sequences maintain their conservation at the three-dimensional structure level. They can play an active role in protein and nucleotide stability, thus not only ensuring proper functioning but also potentiating malfunction and disease. Therefore, insights into any aspect of the repeats – be it structure, function or evolution – would prove to be of some importance. This study aims to address the relationship between protein sequence and its three-dimensional structure, by examining if large cryptic sequence repeats have the same structure.

  6. Thrombin-Binding Aptamer Quadruplex Formation: AFM and Voltammetric Characterization

    Directory of Open Access Journals (Sweden)

    Victor Constantin Diculescu

    2010-01-01

    Full Text Available The adsorption and the redox behaviour of thrombin-binding aptamer (TBA and extended TBA (eTBA were studied using atomic force microscopy and voltammetry at highly oriented pyrolytic graphite and glassy carbon. The different adsorption patterns and degree of surface coverage were correlated with the sequence base composition, presence/absence of K+, and voltammetric behaviour of TBA and eTBA. In the presence of K+, only a few single-stranded sequences present adsorption, while the majority of the molecules forms stable and rigid quadruplexes with no adsorption. Both TBA and eTBA are oxidized and the only anodic peak corresponds to guanine oxidation. Upon addition of K+ ions, TBA and eTBA fold into a quadruplex, causing the decrease of guanine oxidation peak and occurrence of a new peak at a higher potential due to the oxidation of G-quartets. The higher oxidation potential of G-quartets is due to the greater difficulty of electron transfer from the inside of the quadruplex to the electrode surface than electron transfer from the more flexible single strands.

  7. A dual-color flow cytometry protocol for the simultaneous detection of Vibrio parahaemolyticus and Salmonella typhimurium using aptamer conjugated quantum dots as labels

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Nuo; Wu, Shijia [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122 (China); Yu, Ye [Zhangjiagang Entry-Exit Inspection and Quarantine Bureau, Zhangjiangang 215600 (China); Ma, Xiaoyuan; Xia, Yu; Chen, Xiujuan; Huang, Yukun [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122 (China); Wang, Zhouping, E-mail: wangzp@jiangnan.edu.cn [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122 (China)

    2013-12-04

    Graphical abstract: -- Highlights: •Two bacteria were simultaneously detected using QD-apt as labels by flow cytometry. •QD-apt were used for recognition and fluorescence detection of two bacteria. •The method was applied successfully for bacteria detection in real samples. -- Abstract: A sensitive, specific method for the collection and detection of pathogenic bacteria was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with aptamers as the molecular recognition element by flow cytometry. The aptamer sequences were selected using a bacterium-based SELEX strategy in our laboratory for Vibrio parahaemolyticus and Salmonella typhimurium that, when applied in this method, allows for the specific recognition of the bacteria from complex mixtures including shrimp samples. Aptamer-modified QDs (QD-apt) were employed to selectively capture and simultaneously detect the target bacteria with high sensitivity using the fluorescence of the labeled QDs. The signal intensity is amplified due to the high photostability of QDs nanoparticles, resulting in improved sensitivity over methods using individual dye-labeled probes. This proposed method is promising for the sensitive detection of other pathogenic bacteria in food stuff if suitable aptamers are chosen. The method may also provide another potential platform for the application of aptamer-conjugated QDs in flow cytometry.

  8. Aptamers: active targeting ligands for cancer diagnosis and therapy.

    Science.gov (United States)

    Wu, Xu; Chen, Jiao; Wu, Min; Zhao, Julia Xiaojun

    2015-01-01

    Aptamers, including DNA, RNA and peptide aptamers, are a group of promising recognition units that can specifically bind to target molecules and cells. Due to their excellent specificity and high affinity to targets, aptamers have attracted great attention in various fields in which selective recognition units are required. They have been used in biosensing, drug delivery, disease diagnosis and therapy (especially for cancer treatment). In this review, we summarized recent applications of DNA and RNA aptamers in cancer theranostics. The specific binding ability of aptamers to cancer-related markers and cancer cells ensured their high performance for early diagnosis of cancer. Meanwhile, the efficient targeting ability of aptamers to cancer cells and tissues provided a promising way to deliver imaging agents and drugs for cancer imaging and therapy. Furthermore, with the development of nanoscience and nanotechnology, the conjugation of aptamers with functional nanomaterials paved an exciting way for the fabrication of theranostic agents for different types of cancers, which might be a powerful tool for cancer treatment.

  9. Efficient suppression of biofilm formation by a nucleic acid aptamer.

    Science.gov (United States)

    Ning, Yi; Cheng, Lijuan; Ling, Min; Feng, Xinru; Chen, Lingli; Wu, Minxi; Deng, Le

    2015-08-01

    Biofilms are microbial communities that are attached to a solid surface using extracellular polymeric substances. Motility and initial attachment mediated by flagella are required for biofilm formation. Therefore, blocking the motility of flagella is a potential strategy to inhibit biofilm formation. In this study, single-stranded DNA aptamers specific to the Salmonella choleraesuis were selected after 14 cycles of the systematic evolution of ligands by exponential enrichment. Among the selected aptamers, the aptamer 3 showed the highest affinity for S. choleraesuis with a dissociation constant (Kd) of 41 ± 2 nM. Aptamer 3, conjugated with magnetic beads, was then used to capture its binding target on the bacteria. After mass spectrometry and specific binding analysis, the flagellin was identified as the target captured by aptamer 3. Furthermore, inhibition experiments, inverted microscopy and atomic force microscopy demonstrated that aptamer 3 was able to control the biofilm formation and promote the inhibitory effect of an antibiotic on bacterial biofilms. Single-stranded DNA aptamers therefore have great potential as inhibitors of biofilm formation.

  10. Nanostructure shape effects on response of plasmonic aptamer sensors.

    Science.gov (United States)

    Balamurugan, Subramanian; Mayer, Kathryn M; Lee, Seunghyun; Soper, Steven A; Hafner, Jason H; Spivak, David A

    2013-09-01

    A localized surface plasmon resonance (LSPR) sensor surface was fabricated by the deposition of gold nanorods on a glass substrate and subsequent immobilization of the DNA aptamer, which specifically bind to thrombin. This LSPR aptamer sensor showed a response of 6-nm λ(max) shift for protein binding with the detection limit of at least 10 pM, indicating one of the highest sensitivities achieved for thrombin detection by optical extinction LSPR. We also tested the LSPR sensor fabricated using gold bipyramid, which showed higher refractive index sensitivity than the gold nanorods, but the overall response of gold bipyramid sensor appears to be 25% less than that of the gold nanorod substrate, despite the approximately twofold higher refractive index sensitivity. XPS analysis showed that this is due to the low surface density of aptamers on the gold bipyramid compared with gold nanorods. The low surface density of the aptamers on the gold bipyramid surface may be due to the effect of shape of the nanostructure on the kinetics of aptamer monolayer formation. The small size of aptamers relative to other bioreceptors is the key to achieving high sensitivity by biosensors on the basis of LSPR, demonstrated here for protein binding. The generality of aptamer sensors for protein detection using gold nanorod and gold nanobipyramid substrates is anticipated to have a large impact in the important development of sensors toward biomarkers, environmental toxins, and warfare agents.

  11. Structural analysis of DNA sequence: evidence for lateral gene transfer in Thermotoga maritima

    DEFF Research Database (Denmark)

    Worning, Peder; Jensen, Lars Juhl; Nelson, K. E.;

    2000-01-01

    The recently published complete DNA sequence of the bacterium Thermotoga maritima provides evidence, based on protein sequence conservation, for lateral gene transfer between Archaea and Bacteria. We introduce a new method of periodicity analysis of DNA sequences, based on structural parameters......, which brings independent evidence for the lateral gene transfer in the genome of T.maritima, The structural analysis relates the Archaea-like DNA sequences to the genome of Pyrococcus horikoshii. Analysis of 24 complete genomic DNA sequences shows different periodicity patterns for organisms...

  12. FASTR: A novel data format for concomitant representation of RNA sequence and secondary structure information

    Indian Academy of Sciences (India)

    Tungadri Bose; Anirban Dutta; Mohammed Mh; Hemang Gandhi; Sharmila S Mande

    2015-09-01

    Given the importance of RNA secondary structures in defining their biological role, it would be convenient for researchers seeking RNA data if both sequence and structural information pertaining to RNA molecules are made available together. Current nucleotide data repositories archive only RNA sequence data. Furthermore, storage formats which can frugally represent RNA sequence as well as structure data in a single file, are currently unavailable. This article proposes a novel storage format, `FASTR’, for concomitant representation of RNA sequence and structure. The storage efficiency of the proposed FASTR format has been evaluated using RNA data from various microorganisms. Results indicate that the size of FASTR formatted files (containing both RNA sequence as well as structure information) are equivalent to that of FASTA-format files, which contain only RNA sequence information. RNA secondary structure is typically represented using a combination of a string of nucleotide characters along with the corresponding dot-bracket notation indicating structural attributes. `FASTR’ – the novel storage format proposed in the present study enables a frugal representation of both RNA sequence and structural information in the form of a single string. In spite of having a relatively smaller storage footprint, the resultant `fastr’ string(s) retain all sequence as well as secondary structural information that could be stored using a dot-bracket notation. An implementation of the `FASTR’ methodology is available for download at http://metagenomics.atc.tcs.com/compression/fastr.

  13. Biosensor platform based on carbon nanotubes covalently modified with aptamers

    Science.gov (United States)

    Komarov, I. A.; Rubtsova, E. I.; Golovin, A. V.; Bobrinetskiy, I. I.

    2016-12-01

    We developed a new platform for biosensing applications. Aptamers as sensitive agents have a great potential and gives us possibility to have highest possible selectivity among other sensing agents like enzymes or antibodies. We covalently bound aptamers to the functional groups of c-CNTs and then put this system on the surface of polymer substrate. Thus we got high sensitive flexible transparent biological sensors. We also suggest that by varying aptamer type we can make set of biosensors for disease detection which can be integrated into self-healthcare systems and gadgets.

  14. CELL-SELEX: Novel Perspectives of Aptamer-Based Therapeutics

    Directory of Open Access Journals (Sweden)

    Hans P. Wendel

    2008-04-01

    Full Text Available Aptamers, single stranded DNA or RNA molecules, generated by a method called SELEX (systematic evolution of ligands by exponential enrichment have been widely used in various biomedical applications. The newly developed Cell-SELEX (cell based-SELEX targeting whole living cells has raised great expectations for cancer biology, -therapy and regenerative medicine. Combining nanobiotechnology with aptamers, this technology opens the way to more sophisticated applications in molecular diagnosis. This paper gives a review of recent developments in SELEX technologies and new applications of aptamers.

  15. 4SALE – A tool for synchronous RNA sequence and secondary structure alignment and editing

    Directory of Open Access Journals (Sweden)

    Schultz Jörg

    2006-11-01

    Full Text Available Abstract Background In sequence analysis the multiple alignment builds the fundament of all proceeding analyses. Errors in an alignment could strongly influence all succeeding analyses and therefore could lead to wrong predictions. Hand-crafted and hand-improved alignments are necessary and meanwhile good common practice. For RNA sequences often the primary sequence as well as a secondary structure consensus is well known, e.g., the cloverleaf structure of the t-RNA. Recently, some alignment editors are proposed that are able to include and model both kinds of information. However, with the advent of a large amount of reliable RNA sequences together with their solved secondary structures (available from e.g. the ITS2 Database, we are faced with the problem to handle sequences and their associated secondary structures synchronously. Results 4SALE fills this gap. The application allows a fast sequence and synchronous secondary structure alignment for large data sets and for the first time synchronous manual editing of aligned sequences and their secondary structures. This study describes an algorithm for the synchronous alignment of sequences and their associated secondary structures as well as the main features of 4SALE used for further analyses and editing. 4SALE builds an optimal and unique starting point for every RNA sequence and structure analysis. Conclusion 4SALE, which provides an user-friendly and intuitive interface, is a comprehensive toolbox for RNA analysis based on sequence and secondary structure information. The program connects sequence and structure databases like the ITS2 Database to phylogeny programs as for example the CBCAnalyzer. 4SALE is written in JAVA and therefore platform independent. The software is freely available and distributed from the website at http://4sale.bioapps.biozentrum.uni-wuerzburg.de

  16. Sequence, structure, and cooperativity in folding of elementary protein structural motifs.

    Science.gov (United States)

    Lai, Jason K; Kubelka, Ginka S; Kubelka, Jan

    2015-08-11

    Residue-level unfolding of two helix-turn-helix proteins--one naturally occurring and one de novo designed--is reconstructed from multiple sets of site-specific (13)C isotopically edited infrared (IR) and circular dichroism (CD) data using Ising-like statistical-mechanical models. Several model variants are parameterized to test the importance of sequence-specific interactions (approximated by Miyazawa-Jernigan statistical potentials), local structural flexibility (derived from the ensemble of NMR structures), interhelical hydrogen bonds, and native contacts separated by intervening disordered regions (through the Wako-Saitô-Muñoz-Eaton scheme, which disallows such configurations). The models are optimized by directly simulating experimental observables: CD ellipticity at 222 nm for model proteins and their fragments and (13)C-amide I' bands for multiple isotopologues of each protein. We find that data can be quantitatively reproduced by the model that allows two interacting segments flanking a disordered loop (double sequence approximation) and incorporates flexibility in the native contact maps, but neither sequence-specific interactions nor hydrogen bonds are required. The near-identical free energy profiles as a function of the global order parameter are consistent with expected similar folding kinetics for nearly identical structures. However, the predicted folding mechanism for the two motifs is different, reflecting the order of local stability. We introduce free energy profiles for "experimental" reaction coordinates--namely, the degree of local folding as sensed by site-specific (13)C-edited IR, which highlight folding heterogeneity and contrast its overall, average description with the detailed, local picture.

  17. Aptamer-Based Electrochemical Sensing of Lysozyme

    Directory of Open Access Journals (Sweden)

    Alina Vasilescu

    2016-06-01

    Full Text Available Protein analysis and quantification are required daily by thousands of laboratories worldwide for activities ranging from protein characterization to clinical diagnostics. Multiple factors have to be considered when selecting the best detection and quantification assay, including the amount of protein available, its concentration, the presence of interfering molecules, as well as costs and rapidity. This is also the case for lysozyme, a 14.3-kDa protein ubiquitously present in many organisms, that has been identified with a variety of functions: antibacterial activity, a biomarker of several serious medical conditions, a potential allergen in foods or a model of amyloid-type protein aggregation. Since the design of the first lysozyme aptamer in 2001, lysozyme became one of the most intensively-investigated biological target analytes for the design of novel biosensing concepts, particularly with regards to electrochemical aptasensors. In this review, we discuss the state of the art of aptamer-based electrochemical sensing of lysozyme, with emphasis on sensing in serum and real samples.

  18. Graphene- and aptamer-based electrochemical biosensor.

    Science.gov (United States)

    Xu, Ke; Meshik, Xenia; Nichols, Barbara M; Zakar, Eugene; Dutta, Mitra; Stroscio, Michael A

    2014-05-23

    This study investigated the effectiveness of a graphene- and aptamer-based field-effect-transistor-like (FET-like) sensor in detecting lead and potassium ions. The sensor consists of a graphene-covered Si/SiO2 wafer with thrombin binding aptamer (TBA) attached to the graphene layer and terminated by a methylene blue (MB) molecule. K(+) and Pb(2+) both bind to TBA and cause a conformational change, which results in MB moving closer to the graphene surface and donating an electron. Thus, the abundance of K(+) and Pb(2+) can be determined by monitoring the current across the source and drain channel. Device transfer curves were obtained with ambipolar field effect observed. Current readings were taken for K(+) concentrations of 100 μM to 50 mM and Pb(2+) concentrations of 10 μM to 10 mM. As expected, I d decreased as ion concentration increased. In addition, there was a negative shift in V Dirac in response to increased ion concentration.

  19. Structure Prediction and Analysis of Neuraminidase Sequence Variants

    Science.gov (United States)

    Thayer, Kelly M.

    2016-01-01

    Analyzing protein structure has become an integral aspect of understanding systems of biochemical import. The laboratory experiment endeavors to introduce protein folding to ascertain structures of proteins for which the structure is unavailable, as well as to critically evaluate the quality of the prediction obtained. The model system used is the…

  20. Aptamer based electrochemical sensors for emerging environmental pollutants

    Directory of Open Access Journals (Sweden)

    Akhtar eHAYAT

    2014-06-01

    Full Text Available Environmental contaminants monitoring is one of the key issues in understanding and managing hazards to human health and ecosystems. In this context, aptamer based electrochemical sensors have achieved intense significance because of their capability to resolve a potentially large number of problems and challenges in environmental contamination. An aptasensor is a compact analytical device incorporating an aptamer (oligonulceotide as the sensing element either integrated within or intimately associated with a physiochemical transducer surface. Nucleic acid is well known for the function of carrying and passing genetic information, however, it has found a key role in analytical monitoring during recent years. Aptamer based sensors represent a novelty in environmental analytical science and there are great expectations for their promising performance as alternative to conventional analytical tools. This review paper focuses on the recent advances in the development of aptamer based electrochemical sensors for environmental applications with special emphasis on emerging pollutants.

  1. Aptamer Binding Studies Using MicroScale Thermophoresis.

    Science.gov (United States)

    Breitsprecher, Dennis; Schlinck, Nina; Witte, David; Duhr, Stefan; Baaske, Philipp; Schubert, Thomas

    2016-01-01

    The characterization and development of highly specific aptamers requires the analysis of the interaction strength between aptamer and target. MicroScale Thermophoresis (MST) is a rapid and precise method to quantify biomolecular interactions in solution at microliter scale. The basis of this technology is a physical effect referred to as thermophoresis, which describes the directed movement of molecules through temperature gradients. The thermophoretic properties of a molecule depend on its size, charge, and hydration shell. Since at least one of these parameters is altered upon binding of a ligand, this method can be used to analyze virtually any biomolecular interaction in any buffer or complex bioliquid. This section provides a detailed protocol describing how MST is used to obtain quantitative binding parameters for aptamer-target interactions. The two DNA-aptamers HD1 and HD22, which are targeted against human thrombin, are used as model systems to demonstrate a rapid and straightforward screening approach to determine optimal buffer conditions.

  2. Aptamer based electrochemical sensors for emerging environmental pollutants

    Science.gov (United States)

    Hayat, Akhtar; Marty, Jean Louis

    2014-06-01

    Environmental contaminants monitoring is one of the key issues in understanding and managing hazards to human health and ecosystems. In this context, aptamer based electrochemical sensors have achieved intense significance because of their capability to resolve a potentially large number of problems and challenges in environmental contamination. An aptasensor is a compact analytical device incorporating an aptamer (oligonulceotide) as the sensing element either integrated within or intimately associated with a physiochemical transducer surface. Nucleic acid is well known for the function of carrying and passing genetic information, however, it has found a key role in analytical monitoring during recent years. Aptamer based sensors represent a novelty in environmental analytical science and there are great expectations for their promising performance as alternative to conventional analytical tools. This review paper focuses on the recent advances in the development of aptamer based electrochemical sensors for environmental applications with special emphasis on emerging pollutants.

  3. Structure and Evolution of Pre-Main Sequence Stars

    CERN Document Server

    Schulz, Norbert S; Bautz, Mark W; Canizares, Claude C; Davis, John; Dewey, Dan; Huenemoerder, David P; Heilmann, Ralf; Houck, John; Marshall, Herman L; Nowak, Mike; Schattenburg, Mark; Audard, Marc; Drake, Jeremy; Gagne, Marc; Kastner, Joel; Kallman, Tim; Lautenegger, Maurice; Lee, Julia; Miller, Jon; Montmerle, Thierry; Mukai, Koji; Osten, Rachel; Parerels, Frits; Pollock, Andy; Preibisch, Thomas; Raymond, John; Reale, Fabio; Smith, Randall; Testa, Paola; Weintraub, David

    2009-01-01

    Low-mass pre-main sequence (PMS) stars are strong and variable X-ray emitters, as has been well established by EINSTEIN and ROSAT observatories. It was originally believed that this emission was of thermal nature and primarily originated from coronal activity (magnetically confined loops, in analogy with Solar activity) on contracting young stars. Broadband spectral analysis showed that the emission was not isothermal and that elemental abundances were non-Solar. The resolving power of the Chandra and XMM X-ray gratings spectrometers have provided the first, tantalizing details concerning the physical conditions such as temperatures, densities, and abundances that characterize the X-ray emitting regions of young star. These existing high resolution spectrometers, however, simply do not have the effective area to measure diagnostic lines for a large number of PMS stars over required to answer global questions such as: how does magnetic activity in PMS stars differ from that of main sequence stars, how do they ...

  4. Structural conservation of a short, functional, peptide-sequence motif

    OpenAIRE

    Fox-Erlich, Susan; Schiller, Martin R; Gryk, Michael R.

    2009-01-01

    Full length, eukaryotic proteins generally consist of several autonomously folding and functioning domains. Many of these domains are known to function by binding and/or modifying other partner proteins based on the recognition of a short, linear amino sequence contained within the target protein. This article reviews the many bioinformatic tools and resources which discover, define and catalogue the various, known protein domains as well as assist users by identifying domain signatures withi...

  5. APTAMER-BASED SERRS SENSOR FOR THROMBIN DETECTION

    Energy Technology Data Exchange (ETDEWEB)

    Cho, H; Baker, B R; Wachsmann-Hogiu, S; Pagba, C V; Laurence, T A; Lane, S M; Lee, L P; Tok, J B

    2008-07-02

    We describe an aptamer-based Surface Enhanced Resonance Raman Scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human a-thrombin. The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other protein analytes. The specific recognition and binding of thrombin by the thrombin binding aptamer is essential to the mechanism of the aptamer-based sensor, as shown through measurements using negative control oligonucleotides. In addition, the sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, demonstrating that the immobilized, 5{prime}-capped, 3{prime}-capped aptamer is sufficiently robust for clinical diagnostic applications. Furthermore, the proposed sensor may be implemented for multiplexed detection using different aptamer-Raman probe complexes.

  6. Methods To Identify Aptamers against Cell Surface Biomarkers

    Directory of Open Access Journals (Sweden)

    Frédéric Ducongé

    2011-09-01

    Full Text Available Aptamers are nucleic acid-based ligands identified through a process of molecular evolution named SELEX (Systematic Evolution of Ligands by Exponential enrichment. During the last 10-15 years, numerous aptamers have been developed specifically against targets present on or associated with the surface of human cells or infectious pathogens such as viruses, bacteria, fungi or parasites. Several of the aptamers have been described as potent probes, rivalling antibodies, for use in flow cytometry or microscopy. Some have also been used as drugs by inhibiting or activating functions of their targets in a manner similar to neutralizing or agonistic antibodies. Additionally, it is straightforward to conjugate aptamers to other agents without losing their affinity and they have successfully been used in vitro and in vivo to deliver drugs, siRNA, nanoparticles or contrast agents to target cells. Hence, aptamers identified against cell surface biomarkers represent a promising class of ligands. This review presents the different strategies of SELEX that have been developed to identify aptamers for cell surface-associated proteins as well as some of the methods that are used to study their binding on living cells.

  7. Aptamer-targeted RNAi for HIV-1 therapy.

    Science.gov (United States)

    Zhou, Jiehua; Rossi, John J

    2011-01-01

    The highly specific mechanism of RNA (RNAi) that inhibits the expression of disease genes is increasingly being harnessed to develop a new class of therapeutics for a wide variety of human maladies. The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. Herein, we demonstrate novel cell type-specific dual inhibitory function anti-gp120 aptamer-siRNA delivery systems for HIV-1 therapy, in which both the aptamer and the siRNA portions have potent anti-HIV activities. The envelope glycoprotein is expressed on the surface of HIV-1 infected cells, allowing binding and internalization of the aptamer-siRNA chimeric molecules. The Dicer substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells. Our results provide a set of novel aptamer-targeted RNAi therapeutics to combat HIV and further validate the use of anti-gp120 aptamers for delivery of Dicer substrate siRNAs.

  8. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

    Science.gov (United States)

    Kim, Jinho; Olsen, Timothy R.; Zhu, Jing; Hilton, John P.; Yang, Kyung-Ae; Pei, Renjun; Stojanovic, Milan N.; Lin, Qiao

    2016-05-01

    We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling highly efficient isolation of aptamers in drastically reduced times and with minimized consumption of biological material. The approach as such also offers broad target applicability by allowing selection of aptamers with respect to targets that are either surface-immobilized or solution-borne, potentially allowing aptamers to be developed as readily available affinity reagents for a wide range of targets. We demonstrate the utility of this approach on two different procedures, respectively for isolating aptamers against a surface-immobilized protein (immunoglobulin E) and a solution-phase small molecule (bisboronic acid in the presence of glucose). In both cases aptamer candidates were isolated in three rounds of SELEX within a total process time of approximately 10 hours.

  9. Predicting RNA secondary structures from sequence and probing data.

    Science.gov (United States)

    Lorenz, Ronny; Wolfinger, Michael T; Tanzer, Andrea; Hofacker, Ivo L

    2016-07-01

    RNA secondary structures have proven essential for understanding the regulatory functions performed by RNA such as microRNAs, bacterial small RNAs, or riboswitches. This success is in part due to the availability of efficient computational methods for predicting RNA secondary structures. Recent advances focus on dealing with the inherent uncertainty of prediction by considering the ensemble of possible structures rather than the single most stable one. Moreover, the advent of high-throughput structural probing has spurred the development of computational methods that incorporate such experimental data as auxiliary information.

  10. Using structure to explore the sequence alignment space of remote homologs.

    Directory of Open Access Journals (Sweden)

    Andrew Kuziemko

    2011-10-01

    Full Text Available Protein structure modeling by homology requires an accurate sequence alignment between the query protein and its structural template. However, sequence alignment methods based on dynamic programming (DP are typically unable to generate accurate alignments for remote sequence homologs, thus limiting the applicability of modeling methods. A central problem is that the alignment that is "optimal" in terms of the DP score does not necessarily correspond to the alignment that produces the most accurate structural model. That is, the correct alignment based on structural superposition will generally have a lower score than the optimal alignment obtained from sequence. Variations of the DP algorithm have been developed that generate alternative alignments that are "suboptimal" in terms of the DP score, but these still encounter difficulties in detecting the correct structural alignment. We present here a new alternative sequence alignment method that relies heavily on the structure of the template. By initially aligning the query sequence to individual fragments in secondary structure elements and combining high-scoring fragments that pass basic tests for "modelability", we can generate accurate alignments within a small ensemble. Our results suggest that the set of sequences that can currently be modeled by homology can be greatly extended.

  11. Impedance biosensor for the rapid detection of Listeria spp. based on aptamer functionalized Pt-interdigitated microelectrodes array

    Science.gov (United States)

    Sidhu, R.; Rong, Y.; Vanegas, D. C.; Claussen, J.; McLamore, E. S.; Gomes, C.

    2016-05-01

    Listeria monocytogenes is one of the most common causes of food illness deaths worldwide, with multiple outbreaks in the United States alone. Current methods to detect foodborne pathogens are laborious and can take several hours to days to produce results. Thus, faster techniques are needed to detect bacteria within the same reliability level as traditional techniques. This study reports on a rapid, accurate, and sensitive aptamer biosensor device for Listeria spp. detection based on platinum interdigitated array microelectrodes (Pt-IDEs). Pt-IDEs with different geometric electrode gaps were fabricated by lithographic techniques and characterized by cyclic voltammetric (CV), electrochemical impedance spectroscopy (EIS), and potential amperometry (DCPA) measurements of reversible redox species. Based on these results, 50 μm Pt-IDE was chosen to further functionalize with a Listeria monocytogenes DNA aptamer selective to the cell surface protein internalin A, via metal-thiol self-assembly at the 5' end of the 47-mer's. EIS analysis was used to detect Listeria spp. without the need for label amplification and pre-concentration steps. The optimized aptamer concentration of 800 nM was selected to capture the bacteria through internalin A binding and the aptamer hairpin structure near the 3' end. The aptasensor was capable of detecting a wide range of bacteria concentration from 10 to 106 CFU/mL at lower detection limit of 5.39 +/- 0.21 CFU/mL with sensitivity of 268.1 +/- 25.40 (Ohms/log [CFU/mL]) in 17 min. The aptamer based biosensor offers a portable, rapid and sensitive alternative for food safety applications with one of the lowest detection limits reported to date.

  12. Evaluation of medicine effects on the interaction of myoglobin and its aptamer or antibody using atomic force microscopy.

    Science.gov (United States)

    Wang, Qing; Liu, Lin; Yang, Xiaohai; Wang, Kemin; Chen, Nandi; Zhou, Chenchen; Luo, Bianxia; Du, Shasha

    2015-02-17

    The effects of medicine on the biomolecular interaction have been given increasing attention in biochemistry and affinity-based analytics since the environment in vivo is complex especially for the patients. Herein, myoglobin, a biomarker of acute myocardial infarction, was used as a model, and the medicine effects on the interactions of myoglobin/aptamer and myoglobin/antibody were systematically investigated using atomic force microscopy (AFM) for the first time. The results showed that the average binding force and the binding probability of myoglobin/aptamer almost remained unchanged after myoglobin-modified gold substrate was incubated with promazine, amoxicillin, aspirin, and sodium penicillin, respectively. These parameters were changed for myoglobin/antibody after the myoglobin-modified gold substrate was treated with these medicines. For promazine and amoxicillin, they resulted in the change of binding force distribution of myoglobin/antibody (i.e., from unimodal distribution to bimodal distribution) and the increase of binding probability; for aspirin, it only resulted in the change of the binding force distribution, and for sodium penicillin, it resulted in the increase of the average binding force and the binding probability. These results may be attributed to the different interaction modes and binding sites between myoglobin/aptamer and myoglobin/antibody, the different structures between aptamer and antibody, and the effects of medicines on the conformations of myoglobin. These findings could enrich our understanding of medicine effects on the interactions of aptamer and antibody to their target proteins. Moreover, this work will lay a good foundation for better research and extensive applications of biomolecular interaction, especially in the design of biosensors in complex systems.

  13. Molecule-binding dependent assembly of split aptamer and γ-cyclodextrin: A sensitive excimer signaling approach for aptamer biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Fen [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Hubei Key Laboratory of Mine Environmental Pollution Control and Remediation, Environmental Science and Engineering College, Hubei Polytechnic University, Huangshi 435003 (China); Lian, Yan; Li, Jishan; Zheng, Jing; Hu, Yaping; Liu, Jinhua; Huang, Jin [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Yang, Ronghua, E-mail: Yangrh@pku.edu.cn [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China)

    2013-10-17

    Graphical abstract: Adenosine-binding aptamer was splitted into two fragments P2 and P3 which labeled pyrene molecules, mainly produce monomer signal. γ-CD cavity brings P2 and P3 in close proximity, allowing for weak excimer emission. In the presence of target, P2 and P3 are expected to bind ATP and form an aptamer/target complex, leads to large increase of the pyrene excimer fluorescence. -- Highlights: •We assembled split aptamer and γ-cyclodextrin fluorescence biosensors for ATP detection. •The biosensor increased quantum yield and emission lifetime of the excimer. •Time-resolved fluorescence is effective for ATP assay in complicated environment. -- Abstract: A highly sensitive and selective fluorescence aptamer biosensors for the determination of adenosine triphosphate (ATP) was developed. Binding of a target with splitting aptamers labeled with pyrene molecules form stable pyrene dimer in the γ-cyclodextrin (γ-CD) cavity, yielding a strong excimer emission. We have found that inclusion of pyrene dimer in γ-cyclodextrin cavity not only exhibits additive increases in quantum yield and emission lifetime of the excimer, but also facilitates target-induced fusion of the splitting aptamers to form the aptamer/target complex. As proof-of-principle, the approach was applied to fluorescence detection of adenosine triphosphate. With an anti-ATP aptamer, the approach exhibits excimer fluorescence response toward ATP with a maximum signal-to-background ratio of 32.1 and remarkably low detection limit of 80 nM ATP in buffer solution. Moreover, due to the additive fluorescence lifetime of excimer induced by γ-cyclodextrin, time-resolved measurements could be conveniently used to detect as low as 0.5 μM ATP in blood serum quantitatively.

  14. The Study of Correlation Structures of DNA Sequences A Critical Review

    CERN Document Server

    Li, W

    1997-01-01

    The study of correlation structure in the primary sequences of DNA is reviewed. The issues reviewed include: symmetries among 16 base-base correlation functions, accurate estimation of correlation measures, the relationship between $1/f$ and Lorentzian spectra, heterogeneity in DNA sequences, different modeling strategies of the correlation structure of DNA sequences, the difference of correlation structure between coding and non-coding regions (besides the period-3 pattern), and source of broad distribution of domain sizes. Although some of the results remain controversial, a body of work on this topic constitutes a good starting point for future studies.

  15. RNA-Pareto: interactive analysis of Pareto-optimal RNA sequence-structure alignments.

    Science.gov (United States)

    Schnattinger, Thomas; Schöning, Uwe; Marchfelder, Anita; Kestler, Hans A

    2013-12-01

    Incorporating secondary structure information into the alignment process improves the quality of RNA sequence alignments. Instead of using fixed weighting parameters, sequence and structure components can be treated as different objectives and optimized simultaneously. The result is not a single, but a Pareto-set of equally optimal solutions, which all represent different possible weighting parameters. We now provide the interactive graphical software tool RNA-Pareto, which allows a direct inspection of all feasible results to the pairwise RNA sequence-structure alignment problem and greatly facilitates the exploration of the optimal solution set.

  16. A microfluidic surface enhanced Raman spectroscopic biosensor using aptamer functionalized nanopillars

    DEFF Research Database (Denmark)

    Yang, J.; Palla, M.; Bosco, F. G.

    2013-01-01

    This paper presents a microchip incorporating an aptamer-functionalized nanopillar substrate, enabling the specific detection of low-abundance biomolecules using surface enhanced Raman spectroscopy (SERS). In a temperature controlled microchamber, aptamers immobilized on the nanostructure surface...

  17. Comparison of a preQ1 riboswitch aptamer in metabolite-bound and free states with implications for gene regulation.

    Science.gov (United States)

    Jenkins, Jermaine L; Krucinska, Jolanta; McCarty, Reid M; Bandarian, Vahe; Wedekind, Joseph E

    2011-07-15

    Riboswitches are RNA regulatory elements that govern gene expression by recognition of small molecule ligands via a high affinity aptamer domain. Molecular recognition can lead to active or attenuated gene expression states by controlling accessibility to mRNA signals necessary for transcription or translation. Key areas of inquiry focus on how an aptamer attains specificity for its effector, the extent to which the aptamer folds prior to encountering its ligand, and how ligand binding alters expression signal accessibility. Here we present crystal structures of the preQ(1) riboswitch from Thermoanaerobacter tengcongensis in the preQ(1)-bound and free states. Although the mode of preQ(1) recognition is similar to that observed for preQ(0), surface plasmon resonance revealed an apparent K(D) of 2.1 ± 0.3 nm for preQ(1) but a value of 35.1 ± 6.1 nm for preQ(0). This difference can be accounted for by interactions between the preQ(1) methylamine and base G5 of the aptamer. To explore conformational states in the absence of metabolite, the free-state aptamer structure was determined. A14 from the ceiling of the ligand pocket shifts into the preQ(1)-binding site, resulting in "closed" access to the metabolite while simultaneously increasing exposure of the ribosome-binding site. Solution scattering data suggest that the free-state aptamer is compact, but the "closed" free-state crystal structure is inadequate to describe the solution scattering data. These observations are distinct from transcriptional preQ(1) riboswitches of the same class that exhibit strictly ligand-dependent folding. Implications for gene regulation are discussed.

  18. Aptamer-initiated on-particle template-independent enzymatic polymerization (aptamer-OTEP) for electrochemical analysis of tumor biomarkers.

    Science.gov (United States)

    Wang, Pengjuan; Wan, Ying; Deng, Shengyuan; Yang, Shulin; Su, Yan; Fan, Chunhai; Aldalbahi, Ali; Zuo, Xiaolei

    2016-12-15

    Herein, an aptamer-initiated on-particle template-independent enzymatic polymerization (aptamer-OTEP) strategy for electrochemical aptasensor (E-aptasensor) is developed for analysis of cancer biomarker carcino-embryonic antigen (CEA). A pair of DNA aptamers is employed which can be specifically bond with CEA simultaneously. One of the aptamer is thiolated at 3'-terminal and immobilized onto the gold electrode as a capture probe, while the other one has a thiol group at its 5'-terminal and is modified onto the gold nanoparticles surface to form a nanoprobe. In the present of target, the two aptamers can "sandwich" the target, thus the nanoprobe is attached to the electrode. Then terminal deoxynucleotidyl transferase (TdT) is employed to catalyze the incorporation of biotin labeled dNTPs into the 3'-OH terminals of the DNA aptamer on the nanoprobe. The as-generated long DNA oligo tentacles allow specific binding of numerous avidin modified horseradish peroxidase (Av-HRP), resulting in tens of thousands of HRP catalyzed reduction of hydrogen peroxide and sharply increasing electrochemical signals. Taking advantage of the enzyme based nucleic acid amplification and nanoprobe, this strategy is demonstrated to possess the outstanding amplification efficiency.

  19. Allelic diversity and population structure of Bacillus sphaericus as revealed by multilocus sequence typing.

    Science.gov (United States)

    Ge, Yong; Hu, Xiaomin; Zheng, Dasheng; Wu, Yiming; Yuan, Zhiming

    2011-08-01

    The genetic diversity of 35 Bacillus sphaericus strains was analyzed by a newly developed multilocus sequence typing (MLST) scheme, toxin gene pool survey, and mosquito bioassay. The results demonstrated that strains assigned to the same sequence type (ST) had the same occurrence of toxin genes. Further sequence analysis revealed that toxic strains presented a nearly clonal population structure, whereas nontoxic strains had a high level of heterogeneity and were significantly distinct from toxic strains.

  20. Shark myelin basic protein: amino acid sequence, secondary structure, and self-association.

    Science.gov (United States)

    Milne, T J; Atkins, A R; Warren, J A; Auton, W P; Smith, R

    1990-09-01

    Myelin basic protein (MBP) from the Whaler shark (Carcharhinus obscurus) has been purified from acid extracts of a chloroform/methanol pellet from whole brains. The amino acid sequence of the majority of the protein has been determined and compared with the sequences of other MBPs. The shark protein has only 44% homology with the bovine protein, but, in common with other MBPs, it has basic residues distributed throughout the sequence and no extensive segments that are predicted to have an ordered secondary structure in solution. Shark MBP lacks the triproline sequence previously postulated to form a hairpin bend in the molecule. The region containing the putative consensus sequence for encephalitogenicity in the guinea pig contains several substitutions, thus accounting for the lack of activity of the shark protein. Studies of the secondary structure and self-association have shown that shark MBP possesses solution properties similar to those of the bovine protein, despite the extensive differences in primary structure.

  1. Sequence Memory Constraints Give Rise to Language-Like Structure through Iterated Learning

    Science.gov (United States)

    Cornish, Hannah; Dale, Rick; Kirby, Simon; Christiansen, Morten H.

    2017-01-01

    Human language is composed of sequences of reusable elements. The origins of the sequential structure of language is a hotly debated topic in evolutionary linguistics. In this paper, we show that sets of sequences with language-like statistical properties can emerge from a process of cultural evolution under pressure from chunk-based memory constraints. We employ a novel experimental task that is non-linguistic and non-communicative in nature, in which participants are trained on and later asked to recall a set of sequences one-by-one. Recalled sequences from one participant become training data for the next participant. In this way, we simulate cultural evolution in the laboratory. Our results show a cumulative increase in structure, and by comparing this structure to data from existing linguistic corpora, we demonstrate a close parallel between the sets of sequences that emerge in our experiment and those seen in natural language. PMID:28118370

  2. Simultaneous Bayesian estimation of alignment and phylogeny under a joint model of protein sequence and structure.

    Science.gov (United States)

    Herman, Joseph L; Challis, Christopher J; Novák, Ádám; Hein, Jotun; Schmidler, Scott C

    2014-09-01

    For sequences that are highly divergent, there is often insufficient information to infer accurate alignments, and phylogenetic uncertainty may be high. One way to address this issue is to make use of protein structural information, since structures generally diverge more slowly than sequences. In this work, we extend a recently developed stochastic model of pairwise structural evolution to multiple structures on a tree, analytically integrating over ancestral structures to permit efficient likelihood computations under the resulting joint sequence-structure model. We observe that the inclusion of structural information significantly reduces alignment and topology uncertainty, and reduces the number of topology and alignment errors in cases where the true trees and alignments are known. In some cases, the inclusion of structure results in changes to the consensus topology, indicating that structure may contain additional information beyond that which can be obtained from sequences. We use the model to investigate the order of divergence of cytoglobins, myoglobins, and hemoglobins and observe a stabilization of phylogenetic inference: although a sequence-based inference assigns significant posterior probability to several different topologies, the structural model strongly favors one of these over the others and is more robust to the choice of data set.

  3. Implicit Structured Sequence Learning: An FMRI Study of the Structural Mere-Exposure Effect

    Directory of Open Access Journals (Sweden)

    Vasiliki eFolia

    2014-02-01

    Full Text Available In this event-related FMRI study we investigated the effect of five days of implicit acquisition on preference classification by means of an artificial grammar learning (AGL paradigm based on the structural mere-exposure effect and preference classification using a simple right-linear unification grammar. This allowed us to investigate implicit AGL in a proper learning design by including baseline measurements prior to grammar exposure. After 5 days of implicit acquisition, the FMRI results showed activations in a network of brain regions including the inferior frontal (centered on BA 44/45 and the medial prefrontal regions (centered on BA 8/32. Importantly, and central to this study, the inclusion of a naive preference FMRI baseline measurement allowed us to conclude that these FMRI findings were the intrinsic outcomes of the learning process itself and not a reflection of a preexisting functionality recruited during classification, independent of acquisition. Support for the implicit nature of the knowledge utilized during preference classification on day 5 come from the fact that the basal ganglia, associated with implicit procedural learning, were activated during classification, while the medial temporal lobe system, associated with explicit declarative memory, was consistently deactivated. Thus, preference classification in combination with structural mere-exposure can be used to investigate structural sequence processing (syntax in unsupervised AGL paradigms with proper learning designs.

  4. Modified AS1411 Aptamer Suppresses Hepatocellular Carcinoma by Up-Regulating Galectin-14

    OpenAIRE

    Cho, Yuri; Lee, Yun Bin; Lee, Jeong-Hoon; Lee, Dong Hyeon; Cho, Eun Ju; Yu, Su Jong; Kim, Yoon Jun; Kim, Jong In; Im, Jong Hun; Lee, Jung Hwan; Oh, Eun Ju; Yoon, Jung-Hwan

    2016-01-01

    Aptamers are small synthetic oligonucleotides that bind to target proteins with high specificity and affinity. AS1411 is an aptamer that binds to nucleolin, which is overexpressed in the cytoplasm and occurs on the surface of cancer cells. We investigated the therapeutic potential of aptamers in hepatocellular carcinoma (HCC) by evaluating anti-tumor effects and confirming the affinity and specificity of AS1411- and modified AS1411-aptamers in HCC cells. Cell growth was assessed using the MTS...

  5. Large Scale Identification and Categorization of Protein Sequences Using Structured Logistic Regression

    DEFF Research Database (Denmark)

    Pedersen, Bjørn Panella; Ifrim, Georgiana; Liboriussen, Poul

    2014-01-01

    Abstract Background Structured Logistic Regression (SLR) is a newly developed machine learning tool first proposed in the context of text categorization. Current availability of extensive protein sequence databases calls for an automated method to reliably classify sequences and SLR seems well...

  6. Highly sensitive chemiluminescence technology for protein detection using aptamer-based rolling circle amplification platform

    Institute of Scientific and Technical Information of China (English)

    Zhi-Juan Cao; Qian-Wen Peng; Xue Qiu; Cai-Yun Liu; Jian-Zhong Lu

    2011-01-01

    A robust, selective and highly sensitive chemiluminescent (CL) platform for protein assay was presented in this paper. This novel CL approach utilized rolling circle amplification (RCA) as a signal enhancement technique and the 96-well plate as the immobilization and separation carrier. Typically, the antibody immobilized on the surface of 96-well plate was sandwiched with the protein target and the aptamer-primer sequence. This aptamer-primer sequence was then employed as the primer of RCA. Based on this design, a number of the biotinylated probes and streptavidin-horseradish peroxidase (SA-HRP) were captured on the plate, and the CL signal was amplified. In summary, our results demonstrated a robust biosensor with a detection limit of 10 fM that is easy to be established and utilized, and devoid of light source. Therefore, this new technique .will broaden the perspective for future development of DNA-based biosensors for the detection of other protein biomarkers related to clinical diseases, by taking advantages of high sensitivity and selectivity.

  7. seeMotif: exploring and visualizing sequence motifs in 3D structures.

    Science.gov (United States)

    Chang, Darby Tien-Hao; Chien, Ting-Ying; Chen, Chien-Yu

    2009-07-01

    Sequence motifs are important in the study of molecular biology. Motif discovery tools efficiently deliver many function related signatures of proteins and largely facilitate sequence annotation. As increasing numbers of motifs are detected experimentally or predicted computationally, characterizing the functional roles of motifs and identifying the potential synergetic relationships between them are important next steps. A good way to investigate novel motifs is to utilize the abundant 3D structures that have also been accumulated at an astounding rate in recent years. This article reports the development of the web service seeMotif, which provides users with an interactive interface for visualizing sequence motifs on protein structures from the Protein Data Bank (PDB). Researchers can quickly see the locations and conformation of multiple motifs among a number of related structures simultaneously. Considering the fact that PDB sequences are usually shorter than those in sequence databases and/or may have missing residues, seeMotif has two complementary approaches for selecting structures and mapping motifs to protein chains in structures. As more and more structures belonging to previously uncharacterized protein families become available, combining sequence and structure information gives good opportunities to facilitate understanding of protein functions in large-scale genome projects. Available at: http://seemotif.csie.ntu.edu.tw,http://seemotif.ee.ncku.edu.tw or http://seemotif.csbb.ntu.edu.tw.

  8. Sequence Planning for On-Orbit Assembly of Large Space Truss Structures in a Multirobot Environment

    Institute of Scientific and Technical Information of China (English)

    GUO Jifeng; WANG Ping; CUI Naigang

    2006-01-01

    An approach to sequence planning for on-orbit assembly of large space truss structures in a multirobot environment is presented. A hierarchical representation of large space truss structures at the structural volume element level and strut level is adopted. The representation of connectivity matrix and directed graph is respectively presented at the strut level and SVE level. The multirobot environment that consists of autonomous space robots and struts is supposed. Then the multirobot serial assembly strategy, assembly states, assembly tasks and assembly sequences are described. The assembly sequence planning algorithms at the strut level and SVE level are respectively discussed. The results of the simulations show that this approach is feasible and efficient. Two extensions of this approach include more accurate assessment of the efficiency representation and improvements in planning algorithm. In the future, the assembly sequence planning of more large space truss structures and complex multirobot environments and assembly tasks will be considered.

  9. Development of Viral Capsid DNA Aptamer Conjugates as Cell-Targeted Delivery Vehicles

    Science.gov (United States)

    Tong, Gary Jen-Wei

    The ability to generate semi-synthetic DNA-protein conjugates has become increasingly important in the fields of chemical biology and nanobiotechnology. As applications in these fields become more complex, there is also an increased need for methods of attaching synthetic DNA to protein substrates in a well-defined manner. This work outlines the development of new methods for site-specific DNA-protein bioconjugation, as well as the development of novel viral capsid DNA aptamer conjugates for cell-targeting purposes. In order to generate DNA-protein conjugates in a site-specific manner, chemistries orthogonal to native functional groups present on DNA and proteins were exploited. In one method, the attachment of DNA to proteins was achieved via oxime formation. This strategy involved the in situ deprotection of an allyloxycarbonyl-protected alkoxyamine-bearing DNA in the presence of a protein containing a single ketone group. The utility of this approach was demonstrated in the synthesis of a DNA-GFP conjugate. In addition to the oxime formation route, two oxidative coupling methods were also developed for DNA-protein bioconjugation. The first reaction coupled phenylenediamine-containing DNA to anilines, which had been site-specifically incorporated into proteins, in the presence of NaIO4. These reaction conditions were demonstrated on the proteins bacteriophage MS2 and GFP, and were mild enough for the components to retain both protein structure and DNA base-pairing capabilities. The second oxidative coupling reaction conjugated aniline-containing proteins to DNA bearing an o-aminophenol moiety. This reaction occurred under similarly mild conditions; however, higher coupling yields were achieved on MS2 at shorter reaction times by using this strategy. In all three of these methods, the generation of a singly-modified product was achieved. Using one of our oxidative coupling strategies, MS2-DNA aptamer conjugates were synthesized for the development of multivalent

  10. Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures

    DEFF Research Database (Denmark)

    Rangnekar, Abhijit; Zhang, Alex M.; Shiyuan Li, Susan;

    2012-01-01

    Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by...

  11. Can Computationally Designed Protein Sequences Improve Secondary Structure Prediction?

    Science.gov (United States)

    2011-01-01

    ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) U.S. Army...1997) using a significantly larger database of known structures than previously reported in the literature. Methods In this work, the Astral SCOP 1.75

  12. An Algorithm for Finding Conserved Secondary Structure Motifs in Unaligned RNA Sequences

    Institute of Scientific and Technical Information of China (English)

    Giulio Pavesi; Giancarlo Mauri; Graziano Pesole

    2004-01-01

    Several experiments and observations have revealed the fact that small local distinct structural features in RNA molecules are correlated with their biological function, for example, in post-transcriptional regulation of gene expression. Thus, finding similar structural features in a set of RNA sequences known to play the same biological function could provide substantial information concerning which parts of the sequences are responsible for the function itself. Unfortunately, finding common structural elements in RNA molecules is a very challenging task, even if limited to secondary structure. The main difficulty lies in the fact that in nearly all the cases the structure of the molecules is unknown, has to be somehow predicted, and that sequences with little or no similarity can fold into similar structures. Although they differ in some details, the approaches proposed so far are usually based on the preliminary alignment of the sequences and attempt to predict common structures (either local or global, or for some selected regions) for the aligned sequences. These methods give good results when sequence and structure similarity are very high, but function less well when similarity is limited to small and local elements, like single stem-loop motifs. Instead of aligning the sequences, the algorithm we present directly searches for regions of the sequences that can fold into similar structures, where the degree of similarity can be defined by the user. Any information concerning sequence similarity in the motifs can be used either as a search constraint, or a posteriori, by post-processing the output. The search for the regions sharing structural similarity is implemented with the affix tree, a novel text-indexing structure that significantly accelerates the search for patterns having a symmetric layout, such as those forming stem-loop structures. Tests based on experimentally known structures have shown that the algorithm is able to identify functional motifs in

  13. Genome3D: exploiting structure to help users understand their sequences

    OpenAIRE

    Lewis, Tony E.; Sillitoe, Ian; Andreeva, Antonina; Blundell, Tom L.; Buchan, Daniel W. A.; Chothia, Cyrus; Cozzetto, Domenico; Dana, José M.; Filippis, Ioannis; Gough, Julian; Jones, David T.; Kelley, Lawrence A; Kleywegt, Gerard J.; Minneci, Federico; Mistry, Jaina

    2014-01-01

    Genome3D (http://www.genome3d.eu) is a collaborative resource that provides predicted domain annotations and structural models for key sequences. Since introducing Genome3D in a previous NAR paper, we have substantially extended and improved the resource. We have annotated representatives from Pfam families to improve coverage of diverse sequences and added a fast sequence search to the website to allow users to find Genome3D-annotated sequences similar to their own. We have improved and exte...

  14. Using deep RNA sequencing for the structural annotation of the Laccaria bicolor mycorrhizal transcriptome.

    Directory of Open Access Journals (Sweden)

    Peter E Larsen

    Full Text Available BACKGROUND: Accurate structural annotation is important for prediction of function and required for in vitro approaches to characterize or validate the gene expression products. Despite significant efforts in the field, determination of the gene structure from genomic data alone is a challenging and inaccurate process. The ease of acquisition of transcriptomic sequence provides a direct route to identify expressed sequences and determine the correct gene structure. METHODOLOGY: We developed methods to utilize RNA-seq data to correct errors in the structural annotation and extend the boundaries of current gene models using assembly approaches. The methods were validated with a transcriptomic data set derived from the fungus Laccaria bicolor, which develops a mycorrhizal symbiotic association with the roots of many tree species. Our analysis focused on the subset of 1501 gene models that are differentially expressed in the free living vs. mycorrhizal transcriptome and are expected to be important elements related to carbon metabolism, membrane permeability and transport, and intracellular signaling. Of the set of 1501 gene models, 1439 (96% successfully generated modified gene models in which all error flags were successfully resolved and the sequences aligned to the genomic sequence. The remaining 4% (62 gene models either had deviations from transcriptomic data that could not be spanned or generated sequence that did not align to genomic sequence. The outcome of this process is a set of high confidence gene models that can be reliably used for experimental characterization of protein function. CONCLUSIONS: 69% of expressed mycorrhizal JGI "best" gene models deviated from the transcript sequence derived by this method. The transcriptomic sequence enabled correction of a majority of the structural inconsistencies and resulted in a set of validated models for 96% of the mycorrhizal genes. The method described here can be applied to improve gene

  15. Using deep RNA sequencing for the structural annotation of the laccaria bicolor mycorrhizal transcriptome.

    Energy Technology Data Exchange (ETDEWEB)

    Larsen, P. E.; Trivedi, G.; Sreedasyam, A.; Lu, V.; Podila, G. K.; Collart, F. R.; Biosciences Division; Univ. of Alabama

    2010-07-06

    Accurate structural annotation is important for prediction of function and required for in vitro approaches to characterize or validate the gene expression products. Despite significant efforts in the field, determination of the gene structure from genomic data alone is a challenging and inaccurate process. The ease of acquisition of transcriptomic sequence provides a direct route to identify expressed sequences and determine the correct gene structure. We developed methods to utilize RNA-seq data to correct errors in the structural annotation and extend the boundaries of current gene models using assembly approaches. The methods were validated with a transcriptomic data set derived from the fungus Laccaria bicolor, which develops a mycorrhizal symbiotic association with the roots of many tree species. Our analysis focused on the subset of 1501 gene models that are differentially expressed in the free living vs. mycorrhizal transcriptome and are expected to be important elements related to carbon metabolism, membrane permeability and transport, and intracellular signaling. Of the set of 1501 gene models, 1439 (96%) successfully generated modified gene models in which all error flags were successfully resolved and the sequences aligned to the genomic sequence. The remaining 4% (62 gene models) either had deviations from transcriptomic data that could not be spanned or generated sequence that did not align to genomic sequence. The outcome of this process is a set of high confidence gene models that can be reliably used for experimental characterization of protein function. 69% of expressed mycorrhizal JGI 'best' gene models deviated from the transcript sequence derived by this method. The transcriptomic sequence enabled correction of a majority of the structural inconsistencies and resulted in a set of validated models for 96% of the mycorrhizal genes. The method described here can be applied to improve gene structural annotation in other species, provided

  16. Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins.

    Directory of Open Access Journals (Sweden)

    Ran Zichel

    Full Text Available Therapeutic proteins are derived from complex expression/production systems, which can result in minor conformational changes due to preferential codon usage in different organisms, post-translational modifications, etc. Subtle conformational differences are often undetectable by bioanalytical methods but can sometimes profoundly impact the safety, efficacy and stability of products. Numerous bioanalytical methods exist to characterize the primary structure of proteins, post translational modifications; protein-substrate/protein/protein interactions and functional bioassays are available for most proteins that are developed as products. There are however few analytical techniques to detect changes in the tertiary structure of proteins suitable for use during drug development and quality control. For example, x-ray crystallography and NMR are impractical for routine use and do not capture the heterogeneity of the product. Conformation-sensitive antibodies can be used to map proteins. However the development of antibodies to represent sufficient epitopes can be challenging. Other limitations of antibodies include limited supply, high costs, heterogeneity and batch to batch variations in titer. Here we provide proof-of-principle that DNA aptamers to thrombin can be used as surrogate antibodies to characterize conformational changes. We show that aptamers can be used in assays using either an ELISA or a label-free platform to characterize different thrombin products. In addition we replicated a heat-treatment procedure that has previously been shown to not affect protein activity but can result in conformational changes that have serious adverse consequences. We demonstrate that a panel of aptamers (but not an antibody can detect changes in the proteins even when specific activity is unaffected. Our results indicate a novel approach to monitor even small changes in the conformation of proteins which can be used in a routine drug-development and

  17. MODexplorer: an integrated tool for exploring protein sequence, structure and function relationships.

    KAUST Repository

    Kosinski, Jan

    2013-02-08

    SUMMARY: MODexplorer is an integrated tool aimed at exploring the sequence, structural and functional diversity in protein families useful in homology modeling and in analyzing protein families in general. It takes as input either the sequence or the structure of a protein and provides alignments with its homologs along with a variety of structural and functional annotations through an interactive interface. The annotations include sequence conservation, similarity scores, ligand-, DNA- and RNA-binding sites, secondary structure, disorder, crystallographic structure resolution and quality scores of models implied by the alignments to the homologs of known structure. MODexplorer can be used to analyze sequence and structural conservation among the structures of similar proteins, to find structures of homologs solved in different conformational state or with different ligands and to transfer functional annotations. Furthermore, if the structure of the query is not known, MODexplorer can be used to select the modeling templates taking all this information into account and to build a comparative model. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://modorama.biocomputing.it/modexplorer. Website implemented in HTML and JavaScript with all major browsers supported. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  18. Secondary Structure Predictions for Long RNA Sequences Based on Inversion Excursions and MapReduce.

    Science.gov (United States)

    Yehdego, Daniel T; Zhang, Boyu; Kodimala, Vikram K R; Johnson, Kyle L; Taufer, Michela; Leung, Ming-Ying

    2013-05-01

    Secondary structures of ribonucleic acid (RNA) molecules play important roles in many biological processes including gene expression and regulation. Experimental observations and computing limitations suggest that we can approach the secondary structure prediction problem for long RNA sequences by segmenting them into shorter chunks, predicting the secondary structures of each chunk individually using existing prediction programs, and then assembling the results to give the structure of the original sequence. The selection of cutting points is a crucial component of the segmenting step. Noting that stem-loops and pseudoknots always contain an inversion, i.e., a stretch of nucleotides followed closely by its inverse complementary sequence, we developed two cutting methods for segmenting long RNA sequences based on inversion excursions: the centered and optimized method. Each step of searching for inversions, chunking, and predictions can be performed in parallel. In this paper we use a MapReduce framework, i.e., Hadoop, to extensively explore meaningful inversion stem lengths and gap sizes for the segmentation and identify correlations between chunking methods and prediction accuracy. We show that for a set of long RNA sequences in the RFAM database, whose secondary structures are known to contain pseudoknots, our approach predicts secondary structures more accurately than methods that do not segment the sequence, when the latter predictions are possible computationally. We also show that, as sequences exceed certain lengths, some programs cannot computationally predict pseudoknots while our chunking methods can. Overall, our predicted structures still retain the accuracy level of the original prediction programs when compared with known experimental secondary structure.

  19. DNA aptamers as molecular probes for colorectal cancer study.

    Directory of Open Access Journals (Sweden)

    Kwame Sefah

    Full Text Available BACKGROUND: Understanding the molecular features of specific tumors can increase our knowledge about the mechanism(s underlying disease development and progression. This is particularly significant for colorectal cancer, which is a heterogeneous complex of diseases developed in a sequential manner through a multistep carcinogenic process. As such, it is likely that tumors with similar characteristics might originate in the same manner and have a similar molecular behavior. Therefore, specific mapping of the molecular features can be potentially useful for both tumor classification and the development of appropriate therapeutic regimens. However, this can only be accomplished by developing high-affinity molecular probes with the ability to recognize specific markers associated with different tumors. Aptamers can most easily meet this challenge based on their target diversity, flexible manipulation and ease of development. METHODOLOGY AND RESULTS: Using a method known as cell-based Systematic Evolution of Ligands by Exponential enrichment (cell-SELEX and colorectal cancer cultured cell lines DLD-1 and HCT 116, we selected a panel of target-specific aptamers. Binding studies by flow cytometry and confocal microscopy showed that these aptamers have high affinity and selectivity. Our data further show that these aptamers neither recognize normal colon cells (cultured and fresh, nor do they recognize most other cancer cell lines tested. CONCLUSION/SIGNIFICANCE: The selected aptamers can identify specific biomarkers associated with colorectal cancers. We believe that these probes could be further developed for early disease detection, as well as prognostic markers, of colorectal cancers.

  20. Aptamer-Based Technologies in Foodborne Pathogen Detection

    Science.gov (United States)

    Teng, Jun; Yuan, Fang; Ye, Yingwang; Zheng, Lei; Yao, Li; Xue, Feng; Chen, Wei; Li, Baoguang

    2016-01-01

    Aptamers are single stranded DNA or RNA ligands, which can be selected by a method called systematic evolution of ligands by exponential enrichment (SELEX); and they can specifically recognize and bind to their targets. These unique characteristics of aptamers offer great potentials in applications such as pathogen detection and biomolecular screening. Pathogen detection is the critical means in detecting and identifying the problems related to public health and food safety; and only the rapid, sensitive and efficient detection technologies can enable the users to make the accurate assessments on the risks of infections (humans and animals) or contaminations (foods and other commodities) caused by various pathogens. This article reviews the development in the field of the aptamer-based approaches for pathogen detection, including whole-cell SELEX and Genomic SELEX. Nowadays, a variety of aptamer-based biosensors have been developed for pathogen detection. Thus, in this review, we also cover the development in aptamer-based biosensors including optical biosensors for multiple pathogen detection by multiple-labeling or label-free models such as fluorescence detection and surface plasmon resonance, electrochemical biosensors and lateral chromatography test strips, and their applications in pathogen detection and biomolecular screening. While notable progress has been made in the field in the last decade, challenges or drawbacks in their applications such as pathogen detection and biomolecular screening remain to be overcome. PMID:27672383

  1. Label-free selection of RNA aptamers for metabolic engineering.

    Science.gov (United States)

    Hwang, Chuhern; Carothers, James M

    2016-08-15

    RNA aptamers can be assembled into genetic regulatory devices that sense and respond to levels of specific cellular metabolites and thus serve an integral part of designing dynamic control into engineered metabolic pathways. Here, we describe a practical method for generating specific and high affinity aptamers to enable the wider use of in vitro selection and a broader application of aptamers for metabolic engineering. Conventional selection methods involving either radioactive labeling of RNA or the use of label-free methods such as SPR to track aptamer enrichment require resources that are not widely accessible to research groups. We present a label-free selection method that uses small volume spectrophotometers to track RNA enrichment paired with previously characterized affinity chromatography methods. Borrowing techniques used in solid phase peptide synthesis, we present an approach for immobilizing a wide range of metabolites to an amino PEGA matrix. As an illustration, we detail laboratory techniques employed to generate aptamers that bind p-aminophenylalanine, a metabolic precursor for bio-based production of plastics and the pristinamycin family of antibiotics. We focused on the development of methods for ligand immobilization, selection via affinity chromatography, and nucleic acid quantification that can be performed with common laboratory equipment.

  2. Aptamers in Diagnostics and Treatment of Viral Infections

    Directory of Open Access Journals (Sweden)

    Tomasz Wandtke

    2015-02-01

    Full Text Available Aptamers are in vitro selected DNA or RNA molecules that are capable of binding a wide range of nucleic and non-nucleic acid molecules with high affinity and specificity. They have been conducted through the process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment. It serves to reach specificity and considerable affinity to target molecules, including those of viral origin, both proteins and nucleic acids. Properties of aptamers allow detecting virus infected cells or viruses themselves and make them competitive to monoclonal antibodies. Specific aptamers can be used to interfere in each stage of the viral replication cycle and also inhibit its penetration into cells. Many current studies have reported possible application of aptamers as a treatment or diagnostic tool in viral infections, e.g., HIV (Human Immunodeficiency Virus, HBV (Hepatitis B Virus, HCV (Hepatitis C Virus, SARS (Severe Acute Respiratory Syndrome, H5N1 avian influenza and recently spread Ebola. This review presents current developments of using aptamers in the diagnostics and treatment of viral diseases.

  3. Osteomyelitis diagnosis by {sup 99m}Tc radiolabeled aptamers

    Energy Technology Data Exchange (ETDEWEB)

    Santos, S.R.; Ferreira, I.M.; Andrade, A.S.R., E-mail: sararoberta7@hotmail.com, E-mail: imendesf@yahoo.com.br, E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil); Barros, A.L.B.; Cardoso, V.N.; Diniz, O.F., E-mail: brancodebarros@yahoo.com.br, E-mail: valbertcardoso@yahoo.com.br, E-mail: simoneodilia@yahoo.com.br [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Faculdade de Farmacia. Departamento de Analises Clinicas e Toxicologicas

    2015-07-01

    Osteomyelitis, which is characterized by progressive inflammatory destruction and new opposition of bone, is still a difficult infection to treat. The clinical diagnosis in late stages is achieved easily, but an early diagnosis is more challenging. Staphylococcus aureus is a common agent found in osteomyelitis and bone prostheses infection. Diagnosis by scintigraphy has advantages because it is a non-invasive procedure and is able to perform an early diagnosis even before anatomic changes. Thus, nuclear medicine could contribute to an accurate diagnosis since specific radiopharmaceuticals were developed. In this study, aptamers selected to Staphylococcus aureus were labeled with {sup 99m}Tc and used for bacteria identification in an osteomyelitis experimental model. The aptamers selected to S. aureus were directly labelled with {sup 99m}Tc and were evaluated by biodistribution studies. Wistar rats with intraosseous infection in the right paw were used. A random aptamer labelled with {sup 99m}Tc was as control. Six animals were used in each group. The aptamers labeled with {sup 99m}Tc were able to identify the infection foci caused by S. aureus displaying a target/non-target ratio of 2,23 ± 0,20, after 3 h. The control group presented a target/non-target ratio 1,08 ± 0.23. The results indicated that the radiolabeled aptamers were able to identify specifically the infection foci and they should be further explored for infection diagnosis by scintigraphy. (author)

  4. Aptamer-Based Technologies in Foodborne Pathogen Detection

    Directory of Open Access Journals (Sweden)

    Jun Teng

    2016-09-01

    Full Text Available Aptamers are single stranded DNA or RNA ligands, which can be selected by a method called systematic evolution of ligands by exponential enrichment (SELEX; and they can specifically recognize and bind to their targets. These unique characteristics of aptamers offer great potentials in applications such as pathogen detection and biomolecular screening. Pathogen detection is the first and critical means in detecting and identifying the problems related to public health and food safety; and only the rapid, sensitive and efficient detection technologies can enable the users to make to accurate assessments on the risk of infections (humans and animals or contaminations (foods and other commodities caused by various pathogens. This article reviews the developments in the field of the aptamer-based approaches for pathogen detection, including whole-cell SELEX and Genomic SELEX. Nowadays, a variety of aptamer-based biosensors have been developed for pathogen detection. Thus, in this review, we also cover the development of aptamer-based biosensors including optical biosensors for multiple pathogen detection in multiple-labeling or label-free models such as fluorescence detection and surface plasmon resonance, electrochemical biosensors, and lateral chromatography test strips, and their applications in the pathogen detection and biomolecular screening. While notable progress has been made in the field in the last decade, challenges or drawbacks in their applications such as pathogen detection and biomolecular screening, remain to be overcome.

  5. MIPs and Aptamers for Recognition of Proteins in Biomimetic Sensing.

    Science.gov (United States)

    Menger, Marcus; Yarman, Aysu; Erdőssy, Júlia; Yildiz, Huseyin Bekir; Gyurcsányi, Róbert E; Scheller, Frieder W

    2016-07-18

    Biomimetic binders and catalysts have been generated in order to substitute the biological pendants in separation techniques and bioanalysis. The two major approaches use either "evolution in the test tube" of nucleotides for the preparation of aptamers or total chemical synthesis for molecularly imprinted polymers (MIPs). The reproducible production of aptamers is a clear advantage, whilst the preparation of MIPs typically leads to a population of polymers with different binding sites. The realization of binding sites in the total bulk of the MIPs results in a higher binding capacity, however, on the expense of the accessibility and exchange rate. Furthermore, the readout of the bound analyte is easier for aptamers since the integration of signal generating labels is well established. On the other hand, the overall negative charge of the nucleotides makes aptamers prone to non-specific adsorption of positively charged constituents of the sample and the "biological" degradation of non-modified aptamers and ionic strength-dependent changes of conformation may be challenging in some application.

  6. 小分子靶标的核酸适配体筛选的研究进展%Research advances of aptamers selection for small molecule targets

    Institute of Scientific and Technical Information of China (English)

    王勇; 赵新颖; 石冬冬; 杨歌; 屈锋

    2016-01-01

    Aptamers are ribonucleic acid ( RNA ) or single-stranded deoxyribonucleic acid ( ssDNA ) selected by systematic evolution of ligands by exponential enrichment ( SELEX ). Aptamers can identify small molecules,proteins,cells,microorganisms and other targets with high affinity and specificity,and have been widely applied in biology,medicine,food and environmental monitoring. However,available aptamers of practical use are limited. The com-plex and difficult screening of aptamers are the key to restrict its wide application. Differing from biomacromolecules,cells and microorganisms,small molecules have less binding sites and weaker affinity with nucleic acids. And they usually need to be immobilized on substrates. In addition,due to the tiny differences of size,weight and charge of the target-ssDNA/RNA complex and ssDNA/RNA,their separation is difficult. Therefore,the aptamer selection of small molecules is more difficult than biomacromolecules or cells. The selection of methods for immobilizing the targets or library and the optimization of separation process proceed mainly based on the structure characteristics and applications of aptamers. In this paper,the screening methods for molecules with different groups,molecules containing the same group and chiral molecules are introduced. Also,the library design,the methods for separating targets-ssDNA complex and characterizing affinity interaction are discussed. The sequences and dissociation constants(Kd)of about 40 aptamers reported since 2008 are listed.%核酸适配体(aptamer)是通过指数富集配体系统进化(SELEX)技术筛选得到的核糖核酸(RNA)或单链脱氧核糖核酸( ssDNA)。核酸适配体通过高亲和力特异性地识别小分子、蛋白质、细胞、微生物等多种靶标,在生物、医药、食品和环境检测等领域的应用日渐增多。但目前实际可用的核酸适配体有限,其筛选过程复杂,筛选难度大,制约了其应用。与生物

  7. Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2′-C-piperazino-UNA monomer

    DEFF Research Database (Denmark)

    Jensen, Troels B.; Henriksen, Jonas Rosager; Rasmussen, Bjarne E.;

    2011-01-01

    Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2′-C-piperazino-UNA residue and UNA residues incorporated in several positions on th...

  8. A Conjugated Aptamer-Gold Nanoparticle Fluorescent Probe for Highly Sensitive Detection of rHuEPO-α

    Directory of Open Access Journals (Sweden)

    Zhaoyang Zhang

    2011-11-01

    Full Text Available We present here a novel conjugated aptamer-gold nanoparticle (Apt-AuNPs fluorescent probe and its application for specific detection of recombinant human erythropoietin-α (rHuEPO-α. In this nanobiosensor, 12 nm AuNPs function as both a nano-scaffold and a nano-quencher (fluorescent energy acceptor, on the surface of which the complementary sequences are linked (as cODN-AuNPs and pre-hybridized with carboxymethylfluorescein (FAM-labeled anti-rHuEPO-α aptamers. Upon target protein binding, the aptamers can be released from the AuNP surface and the fluorescence signal is restored. Key variables such as the length of linker, the hybridization site and length have been designed and optimized. Full performance evaluation including sensitivity, linear range and interference substances are also described. This nanobiosensor provides a promising approach for a simple and direct quantification of rHuEPO-α concentrations as low as 0.92 nM within a few hours.

  9. Cell-specific RNA aptamer against human CCR5 specifically targets HIV-1 susceptible cells and inhibits HIV-1 infectivity.

    Science.gov (United States)

    Zhou, Jiehua; Satheesan, Sangeetha; Li, Haitang; Weinberg, Marc S; Morris, Kevin V; Burnett, John C; Rossi, John J

    2015-03-19

    The C-C chemokine receptor type 5 (CCR5) is a receptor expressed by T cells and macrophages that serves as a coreceptor for macrophage-tropic HIV-1. Loss of CCR5 is associated with resistance to HIV-1. Here, we combine the live-cell-based SELEX with high-throughput sequencing technology to generate CCR5 RNA aptamers capable of specifically targeting HIV-1 susceptible cells (as small interfering RNA [siRNA] delivery agent) and inhibiting HIV-1 infectivity (as antiviral agent) via block of the CCR5 required for HIV-1 to enter cells. One of the best candidates, G-3, efficiently bound and was internalized into human CCR5-expressing cells. The G-3 specifically neutralized R5 virus infection in primary peripheral blood mononuclear cells, and in vivo generated human CD4(+) T cells with a nanomolar inhibitory concentration 50%. G-3 was also capable of transferring functional siRNAs to CCR5-expressing cells. Collectively, the cell-specific, internalizing, CCR5-targeted aptamers and aptamer-siRNA conjugates offer promise for overcoming some of the current challenges of drug resistance in HIV-1 by providing cell-type- or tissue-specific delivery of various therapeutic moieties.

  10. Investigation of the protein osteocalcin of Camelops hesternus: Sequence, structure and phylogenetic implications

    Science.gov (United States)

    Humpula, James F.; Ostrom, Peggy H.; Gandhi, Hasand; Strahler, John R.; Walker, Angela K.; Stafford, Thomas W.; Smith, James J.; Voorhies, Michael R.; George Corner, R.; Andrews, Phillip C.

    2007-12-01

    Ancient DNA sequences offer an extraordinary opportunity to unravel the evolutionary history of ancient organisms. Protein sequences offer another reservoir of genetic information that has recently become tractable through the application of mass spectrometric techniques. The extent to which ancient protein sequences resolve phylogenetic relationships, however, has not been explored. We determined the osteocalcin amino acid sequence from the bone of an extinct Camelid (21 ka, Camelops hesternus) excavated from Isleta Cave, New Mexico and three bones of extant camelids: bactrian camel ( Camelus bactrianus); dromedary camel ( Camelus dromedarius) and guanaco ( Llama guanacoe) for a diagenetic and phylogenetic assessment. There was no difference in sequence among the four taxa. Structural attributes observed in both modern and ancient osteocalcin include a post-translation modification, Hyp 9, deamidation of Gln 35 and Gln 39, and oxidation of Met 36. Carbamylation of the N-terminus in ancient osteocalcin may result in blockage and explain previous difficulties in sequencing ancient proteins via Edman degradation. A phylogenetic analysis using osteocalcin sequences of 25 vertebrate taxa was conducted to explore osteocalcin protein evolution and the utility of osteocalcin sequences for delineating phylogenetic relationships. The maximum likelihood tree closely reflected generally recognized taxonomic relationships. For example, maximum likelihood analysis recovered rodents, birds and, within hominins, the Homo-Pan-Gorilla trichotomy. Within Artiodactyla, character state analysis showed that a substitution of Pro 4 for His 4 defines the Capra-Ovis clade within Artiodactyla. Homoplasy in our analysis indicated that osteocalcin evolution is not a perfect indicator of species evolution. Limited sequence availability prevented assigning functional significance to sequence changes. Our preliminary analysis of osteocalcin evolution represents an initial step towards a

  11. Identification of 5' capped structure and 3' terminal sequence of hepatitis E virus isolated from Morocco

    Institute of Scientific and Technical Information of China (English)

    Guo-Bing Chen; Ji-Hong Meng

    2004-01-01

    AIM: To examine 5' and 3' terminal sequences of hepatitis E virus (HEV) isolated from Morocco, to confirm 5' methylated cap structure of the genome, and to investigate whether the 3' UTR can be used to distinguish HEV genotypes instead of HEV complete genome sequence.METHODS: RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) was employed to obtain the 5' and 3' terminal sequences of HEV Morocco strain. The 3' UTR sequence of the Morocco strain was compared with that of the other 29 HEV strains using the DNAStar software.RESULTS: The 5' PCR product was obtained only from the RLM-RACE based on the capped RNA template. The 5' UTR of the Morocco strain had 26 nucleotides, and the 3' UTR had 65 nucleotides upstream to the polyA. The 5' UTR between HEV strains had only point mutations of nucleotides.The phylogenetic tree based on the sequences of 3' UTR was not the same as that based on the complete sequences.CONCLUSION: The genome of HEV Morocco strain was methylated cap structure. The 3' terminal sequence can not be used for distinguishing HEV genotype for all HEV strains in place of the whole HEV genome sequence.

  12. Ab initio protein folding simulations using atomic burials as informational intermediates between sequence and structure.

    Science.gov (United States)

    van der Linden, Marx Gomes; Ferreira, Diogo César; de Oliveira, Leandro Cristante; Onuchic, José N; de Araújo, Antônio F Pereira

    2014-07-01

    The three-dimensional structure of proteins is determined by their linear amino acid sequences but decipherment of the underlying protein folding code has remained elusive. Recent studies have suggested that burials, as expressed by atomic distances to the molecular center, are sufficiently informative for structural determination while potentially obtainable from sequences. Here we provide direct evidence for this distinctive role of burials in the folding code, demonstrating that burial propensities estimated from local sequence can indeed be used to fold globular proteins in ab initio simulations. We have used a statistical scheme based on a Hidden Markov Model (HMM) to classify all heavy atoms of a protein into a small number of burial atomic types depending on sequence context. Molecular dynamics simulations were then performed with a potential that forces all atoms of each type towards their predicted burial level, while simple geometric constraints were imposed on covalent structure and hydrogen bond formation. The correct folded conformation was obtained and distinguished in simulations that started from extended chains for a selection of structures comprising all three folding classes and high burial prediction quality. These results demonstrate that atomic burials can act as informational intermediates between sequence and structure, providing a new conceptual framework for improving structural prediction and understanding the fundamentals of protein folding.

  13. Sequence Analysis of the Protein Structure Homology Modeling of Growth Hormone Gene from Salmo trutta caspius

    Directory of Open Access Journals (Sweden)

    Abolhasan Rezaei

    2012-03-01

    Full Text Available In view of the growth hormone protein investigated and characterized from Salmo trutta caspius. Growth hormone gene in the Salmo trutta caspius have six exons in the full length that is translated into a Molecular Weight (kDa: ssDNA: 64.98 and dsDNA: 129.6. There are also 210 amino acid residue. The assembled full length of DNA contains open reading frame of growth hormone gene that contains 15 sequences in the full length. The average GC content is 47% and AT content is 53%. This protein multiple alignment has shown that this peptide is 100% identical to the corresponding homologous protein in the growth hormone protein which including Salmo salar (Accession number: AAA49558.1 and Rainbow trout (Salmo trutta (Accession number: AAA49555.1" sequences. The sequence of protein had deposited in Gene Bank, Accession number: AEK70940. Also we were analyzed second and third structure between sequences reported in Gene Bank Network system. The results are shown, there are homology between second structure in three sequences including: Salmo trutta caspius, Salmo salar and Rainbow trout. Regarding third structure, Salmo trutta caspius and Salmo salar are same type, but Rainbow trout has different homology with Salmo trutta caspius and Salmo salar. However, the sequences were observed three parallel " helix and in second structure there were almost same percent β sheet.

  14. Key Issues in Modeling of Complex 3D Structures from Video Sequences

    Directory of Open Access Journals (Sweden)

    Shengyong Chen

    2012-01-01

    Full Text Available Construction of three-dimensional structures from video sequences has wide applications for intelligent video analysis. This paper summarizes the key issues of the theory and surveys the recent advances in the state of the art. Reconstruction of a scene object from video sequences often takes the basic principle of structure from motion with an uncalibrated camera. This paper lists the typical strategies and summarizes the typical solutions or algorithms for modeling of complex three-dimensional structures. Open difficult problems are also suggested for further study.

  15. Aptamer-based therapeutics: new approaches to combat human viral diseases.

    Science.gov (United States)

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

    2013-11-25

    Viruses replicate inside the cells of an organism and continuously evolve to contend with an ever-changing environment. Many life-threatening diseases, such as AIDS, SARS, hepatitis and some cancers, are caused by viruses. Because viruses have small genome sizes and high mutability, there is currently a lack of and an urgent need for effective treatment for many viral pathogens. One approach that has recently received much attention is aptamer-based therapeutics. Aptamer technology has high target specificity and versatility, i.e., any viral proteins could potentially be targeted. Consequently, new aptamer-based therapeutics have the potential to lead a revolution in the development of anti-infective drugs. Additionally, aptamers can potentially bind any targets and any pathogen that is theoretically amenable to rapid targeting, making aptamers invaluable tools for treating a wide range of diseases. This review will provide a broad, comprehensive overview of viral therapies that use aptamers. The aptamer selection process will be described, followed by an explanation of the potential for treating virus infection by aptamers. Recent progress and prospective use of aptamers against a large variety of human viruses, such as HIV-1, HCV, HBV, SCoV, Rabies virus, HPV, HSV and influenza virus, with particular focus on clinical development of aptamers will also be described. Finally, we will discuss the challenges of advancing antiviral aptamer therapeutics and prospects for future success.

  16. Thousands of corresponding human and mouse genomic regions unalignable in primary sequence contain common RNA structure

    DEFF Research Database (Denmark)

    Torarinsson, Elfar; Sawera, Milena; Havgaard, Jakob Hull;

    2006-01-01

    Human and mouse genome sequences contain roughly 100,000 regions that are unalignable in primary sequence and neighbor corresponding alignable regions between both organisms. These pairs are generally assumed to be nonconserved, although the level of structural conservation between these has never...... been investigated. Owing to the limitations in computational methods, comparative genomics has been lacking the ability to compare such nonconserved sequence regions for conserved structural RNA elements. We have investigated the presence of structural RNA elements by conducting a local structural...... overlapped by transfrags than regions that are not overlapped by transfrags. To verify the coexpression between predicted candidates in human and mouse, we conducted expression studies by RT-PCR and Northern blotting on mouse candidates, which overlap with transfrags on human chromosome 20. RT-PCR results...

  17. Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides.

    Science.gov (United States)

    Soontornworajit, Boonchoy; Zhou, Jing; Snipes, Matthew P; Battig, Mark R; Wang, Yong

    2011-10-01

    Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer--protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer--protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.

  18. Probing the coagulation pathway with aptamers identifies combinations that synergistically inhibit blood clot formation.

    Science.gov (United States)

    Bompiani, Kristin M; Lohrmann, Jens L; Pitoc, George A; Frederiksen, James W; Mackensen, George B; Sullenger, Bruce A

    2014-08-14

    Coordinated enzymatic reactions regulate blood clot generation. To explore the contributions of various coagulation enzymes in this process, we utilized a panel of aptamers against factors VIIa, IXa, Xa, and prothrombin. Each aptamer dose-dependently inhibited clot formation, yet none was able to completely impede this process in highly procoagulant settings. However, several combinations of two aptamers synergistically impaired clot formation. One extremely potent aptamer combination was able to maintain human blood fluidity even during extracorporeal circulation, a highly procoagulant setting encountered during cardiopulmonary bypass surgery. Moreover, this aptamer cocktail could be rapidly reversed with antidotes to restore normal hemostasis, indicating that even highly potent aptamer combinations can be rapidly controlled. These studies highlight the potential utility of using sets of aptamers to probe the functions of proteins in molecular pathways for research and therapeutic ends.

  19. Development of an aptamer-based affinity purification method for vascular endothelial growth factor

    Directory of Open Access Journals (Sweden)

    Maren Lönne

    2015-12-01

    Full Text Available Since aptamers bind their targets with high affinity and specificity, they are promising alternative ligands in protein affinity purification. As aptamers are chemically synthesized oligonucleotides, they can be easily produced in large quantities regarding GMP conditions allowing their application in protein production for therapeutic purposes. Several advantages of aptamers compared to antibodies are described in general within this paper. Here, an aptamer directed against the human Vascular Endothelial Growth Factor (VEGF was used as affinity ligand for establishing a purification platform for VEGF in small scale. The aptamer was covalently immobilized on magnetic beads in a controlled orientation resulting in a functional active affinity matrix. Target binding was optimized by introduction of spacer molecules and variation of aptamer density. Further, salt-induced target elution was demonstrated as well as VEGF purification from a complex protein mixture proving the specificity of protein-aptamer binding.

  20. Progress and Challenges in Developing Aptamer-Functionalized Targeted Drug Delivery Systems

    Directory of Open Access Journals (Sweden)

    Feng Jiang

    2015-10-01

    Full Text Available Aptamers, which can be screened via systematic evolution of ligands by exponential enrichment (SELEX, are superior ligands for molecular recognition due to their high selectivity and affinity. The interest in the use of aptamers as ligands for targeted drug delivery has been increasing due to their unique advantages. Based on their different compositions and preparation methods, aptamer-functionalized targeted drug delivery systems can be divided into two main categories: aptamer-small molecule conjugated systems and aptamer-nanomaterial conjugated systems. In this review, we not only summarize recent progress in aptamer selection and the application of aptamers in these targeted drug delivery systems but also discuss the advantages, challenges and new perspectives associated with these delivery systems.

  1. Progress and Challenges in Developing Aptamer-Functionalized Targeted Drug Delivery Systems.

    Science.gov (United States)

    Jiang, Feng; Liu, Biao; Lu, Jun; Li, Fangfei; Li, Defang; Liang, Chao; Dang, Lei; Liu, Jin; He, Bing; Badshah, Shaikh Atik; Lu, Cheng; He, Xiaojuan; Guo, Baosheng; Zhang, Xiao-Bing; Tan, Weihong; Lu, Aiping; Zhang, Ge

    2015-01-01

    Aptamers, which can be screened via systematic evolution of ligands by exponential enrichment (SELEX), are superior ligands for molecular recognition due to their high selectivity and affinity. The interest in the use of aptamers as ligands for targeted drug delivery has been increasing due to their unique advantages. Based on their different compositions and preparation methods, aptamer-functionalized targeted drug delivery systems can be divided into two main categories: aptamer-small molecule conjugated systems and aptamer-nanomaterial conjugated systems. In this review, we not only summarize recent progress in aptamer selection and the application of aptamers in these targeted drug delivery systems but also discuss the advantages, challenges and new perspectives associated with these delivery systems.

  2. Theoretical modeling of masking DNA application in aptamer-facilitated biomarker discovery.

    Science.gov (United States)

    Cherney, Leonid T; Obrecht, Natalia M; Krylov, Sergey N

    2013-04-16

    In aptamer-facilitated biomarker discovery (AptaBiD), aptamers are selected from a library of random DNA (or RNA) sequences for their ability to specifically bind cell-surface biomarkers. The library is incubated with intact cells, and cell-bound DNA molecules are separated from those unbound and amplified by the polymerase chain reaction (PCR). The partitioning/amplification cycle is repeated multiple times while alternating target cells and control cells. Efficient aptamer selection in AptaBiD relies on the inclusion of masking DNA within the cell and library mixture. Masking DNA lacks primer regions for PCR amplification and is typically taken in excess to the library. The role of masking DNA within the selection mixture is to outcompete any nonspecific binding sequences within the initial library, thus allowing specific DNA sequences (i.e., aptamers) to be selected more efficiently. Efficient AptaBiD requires an optimum ratio of masking DNA to library DNA, at which aptamers still bind specific binding sites but nonaptamers within the library do not bind nonspecific binding sites. Here, we have developed a mathematical model that describes the binding processes taking place within the equilibrium mixture of masking DNA, library DNA, and target cells. An obtained mathematical solution allows one to estimate the concentration of masking DNA that is required to outcompete the library DNA at a desirable ratio of bound masking DNA to bound library DNA. The required concentration depends on concentrations of the library and cells as well as on unknown cell characteristics. These characteristics include the concentration of total binding sites on the cell surface, N, and equilibrium dissociation constants, K(nsL) and K(nsM), for nonspecific binding of the library DNA and masking DNA, respectively. We developed a theory that allows the determination of N, K(nsL), and K(nsM) based on measurements of EC50 values for cells mixed separately with the library and masking DNA

  3. In situ labeling and imaging of cellular protein via a bi-functional anticancer aptamer and its fluorescent ligand.

    Science.gov (United States)

    Ai, Jun; Li, Tao; Li, Bingling; Xu, Yuanhong; Li, Dan; Liu, Zuojia; Wang, Erkang

    2012-09-05

    In this article, we reported a novel approach for in situ labeling and imaging HeLa cancer cells utilizing a bifunctional aptamer (AS1411) and its fluorescent ligand, protoporphyrin IX (PPIX). In the presence of potassium ion, AS1411 folded to G-quadruplex structure, binded fluorescent ligand (PPIX) with fluorescent enhancement, and targeted the nucleolin overexpressed by cancer cells. Consequently, bioimaging of cancer cells specifically were realized by laser scanning confocal microscope. The bioimaging strategy with AS1411-PPIX complex was capable to distinguish HeLa cancer cells from normal cells unambiguously, and fluorescence imaging of cancer cells was also realized in human serum. Moreover, the bioimaging method was very facile, effective and need not any covalent modification. These results illustrated that the useful approach can provide a novel clue for bioimaging based on non-covalent bifunctional aptamer in clinic diagnosis.

  4. Evolutionary evidence for alternative structure in RNA sequence co-variation.

    Directory of Open Access Journals (Sweden)

    Justin Ritz

    Full Text Available Sequence conservation and co-variation of base pairs are hallmarks of structured RNAs. For certain RNAs (e.g. riboswitches, a single sequence must adopt at least two alternative secondary structures to effectively regulate the message. If alternative secondary structures are important to the function of an RNA, we expect to observe evolutionary co-variation supporting multiple conformations. We set out to characterize the evolutionary co-variation supporting alternative conformations in riboswitches to determine the extent to which alternative secondary structures are conserved. We found strong co-variation support for the terminator, P1, and anti-terminator stems in the purine riboswitch by extending alignments to include terminator sequences. When we performed Boltzmann suboptimal sampling on purine riboswitch sequences with terminators we found that these sequences appear to have evolved to favor specific alternative conformations. We extended our analysis of co-variation to classic alignments of group I/II introns, tRNA, and other classes of riboswitches. In a majority of these RNAs, we found evolutionary evidence for alternative conformations that are compatible with the Boltzmann suboptimal ensemble. Our analyses suggest that alternative conformations are selected for and thus likely play functional roles in even the most structured of RNAs.

  5. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  6. Protein secondary structure prediction for a single-sequence using hidden semi-Markov models

    OpenAIRE

    2006-01-01

    Abstract Background The accuracy of protein secondary structure prediction has been improving steadily towards the 88% estimated theoretical limit. There are two types of prediction algorithms: Single-sequence prediction algorithms imply that information about other (homologous) proteins is not available, while algorithms of the second type imply that information about homologous proteins is available, and use it intensively. The single-sequence algorithms could make an important contribution...

  7. A Java applet for multiple linked visualization of protein structure and sequence.

    Science.gov (United States)

    Oldfield, Thomas J

    2004-04-01

    The amount of biological data available from experimental techniques is huge, and rapidly expanding. The ability to make sense of this vast amount of data requires that we make correlations between distinct biological disciplines using visualization techniques to highlight the critical information. This article describes the visualization techniques of dynamic data brushing, view context maintenance, fisheye sequence view, and a magic lens that have been developed to display protein structure and sequence information.

  8. Bioactivity of 2′-deoxyinosine-incorporated aptamer AS1411

    OpenAIRE

    Xinmeng Fan; Lidan Sun; Yun Wu; Lihe Zhang; Zhenjun Yang

    2016-01-01

    Aptamers can be chemically modified to enhance nuclease resistance and increase target affinity. In this study, we performed chemical modification of 2′-deoxyinosine in AS1411, an anti-proliferative G-rich oligodeoxynucleotide aptamer, which binds selectively to the nucleolin protein. Its function was augmented when 2′-deoxyinosine was incorporated at positions 12, 13, 15, and 24 of AS1411, respectively. In addition, double incorporation of 2′-deoxyinosine at positions 12 and 24 (FAN-1224dI),...

  9. Aptamer-Binding Directed DNA Origami Pattern for Logic Gates.

    Science.gov (United States)

    Yang, Jing; Jiang, Shuoxing; Liu, Xiangrong; Pan, Linqiang; Zhang, Cheng

    2016-12-14

    In this study, an aptamer-substrate strategy is introduced to control programmable DNA origami pattern. Combined with DNA aptamer-substrate binding and DNAzyme-cutting, small DNA tiles were specifically controlled to fill into the predesigned DNA origami frame. Here, a set of DNA logic gates (OR, YES, and AND) are performed in response to the stimuli of adenosine triphosphate (ATP) and cocaine. The experimental results are confirmed by AFM imaging and time-dependent fluorescence changes, demonstrating that the geometric patterns are regulated in a controllable and programmable manner. Our approach provides a new platform for engineering programmable origami nanopatterns and constructing complex DNA nanodevices.

  10. Automatic phylogenetic classification of bacterial beta-lactamase sequences including structural and antibiotic substrate preference information.

    Science.gov (United States)

    Ma, Jianmin; Eisenhaber, Frank; Maurer-Stroh, Sebastian

    2013-12-01

    Beta lactams comprise the largest and still most effective group of antibiotics, but bacteria can gain resistance through different beta lactamases that can degrade these antibiotics. We developed a user friendly tree building web server that allows users to assign beta lactamase sequences to their respective molecular classes and subclasses. Further clinically relevant information includes if the gene is typically chromosomal or transferable through plasmids as well as listing the antibiotics which the most closely related reference sequences are known to target and cause resistance against. This web server can automatically build three phylogenetic trees: the first tree with closely related sequences from a Tachyon search against the NCBI nr database, the second tree with curated reference beta lactamase sequences, and the third tree built specifically from substrate binding pocket residues of the curated reference beta lactamase sequences. We show that the latter is better suited to recover antibiotic substrate assignments through nearest neighbor annotation transfer. The users can also choose to build a structural model for the query sequence and view the binding pocket residues of their query relative to other beta lactamases in the sequence alignment as well as in the 3D structure relative to bound antibiotics. This web server is freely available at http://blac.bii.a-star.edu.sg/.

  11. STUDY ON SEQUENCE STRUCTURE OF ACRYLAMIDE-ACRYLATE COPOLYMERS BY 13C-NMR METHOD

    Institute of Scientific and Technical Information of China (English)

    YUAN Dongwu; ZHU Shannong; YANG Xiaozhen

    1987-01-01

    Triad sequence distributions in a series of P(AM/AA) with different AA% were calculated from copolymerization reactivity ratio r1 and r2 based on first order Markov statistic model, and the calculated data compared with observed ones from 13C-NMR spectra showed good agreement with each other, The sequence distribution in P(AM/AA) obtained under our experimental conditions fits in with first order Markov statistic model. A significant sequence structure difference was observed between P(AM/AA) and alkaline hydrolyzed polyacrylamide, ABA triad (acrylate unit center), AAA and AAB triads (acrylamide unit center) dominated in hydrolyzed ones.

  12. Multifunctional aptamer-based nanoparticles for targeted drug delivery to circumvent cancer resistance.

    Science.gov (United States)

    Liu, Juan; Wei, Tuo; Zhao, Jing; Huang, Yuanyu; Deng, Hua; Kumar, Anil; Wang, Chenxuan; Liang, Zicai; Ma, Xiaowei; Liang, Xing-Jie

    2016-06-01

    By its unique advantages over traditional medicine, nanomedicine has offered new strategies for cancer treatment. In particular, the development of drug delivery strategies has focused on nanoscale particles to improve bioavailability. However, many of these nanoparticles are unable to overcome tumor resistance to chemotherapeutic agents. Recently, new opportunities for drug delivery have been provided by oligonucleotides that can self-assemble into three-dimensional nanostructures. In this work, we have designed and developed functional DNA nanostructures to deliver the chemotherapy drug doxorubicin (Dox) to resistant cancer cells. These nanostructures have two components. The first component is a DNA aptamer, which forms a dimeric G-quadruplex nanostructure to target cancer cells by binding with nucleolin. The second component is double-stranded DNA (dsDNA), which is rich in -GC- base pairs that can be applied for Dox delivery. We demonstrated that Dox was able to efficiently intercalate into dsDNA and this intercalation did not affect the aptamer's three-dimensional structure. In addition, the Aptamer-dsDNA (ApS) nanoparticle showed good stability and protected the dsDNA from degradation in bovine serum. More importantly, the ApS&Dox nanoparticle efficiently reversed the resistance of human breast cancer cells to Dox. The mechanism circumventing doxorubicin resistance by ApS&Dox nanoparticles may be predominantly by cell cycle arrest in S phase, effectively increased cell uptake and decreased cell efflux of doxorubicin. Furthermore, the ApS&Dox nanoparticles could effectively inhibit tumor growth, while less cardiotoxicity was observed. Overall, this functional DNA nanostructure provides new insights into the design of nanocarriers to overcome multidrug resistance through targeted drug delivery.

  13. Comparative analysis of MR sequences to detect structural brain lesions in tuberous sclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Pinto Gama, Hugo Pereira; Campos Meirelles, Rogerio Goncalves de; Mendonca do Rego, Jose Iram [Santa Casa de Misericordia de Sao Paulo, Section of Radiology, Sao Paulo (Brazil); Rocha, Antonio Jose da; Silva, Carlos Jorge da [Santa Casa de Misericordia de Sao Paulo, Section of Radiology, Centro de Medicina Diagnostica Fleury, Sao Paulo (Brazil); Braga, Flavio Tulio [Federal University of Sao Paulo, Escola Paulista de Medicina, Section of Radiology, Centro de Medicina Diagnostica Fleury, Santa Casa de Misericordia de Sao Paulo, Department of Diagnostic Imaging, Sao Paulo (Brazil); Martins Maia, Antonio Carlos [Federal University of Sao Paulo, Escola Paulista de Medicina, Section of Radiology, Centro de Medicina Diagnostica Fleury, Department of Neurology, Sao Paulo (Brazil); Lederman, Henrique Manoel [Federal University of Sao Paulo, Escola Paulista de Medicina, Division of Diagnostic Imaging in Pediatrics, Department of Diagnostic Imaging, Sao Paulo (Brazil)

    2006-02-01

    Tuberous sclerosis (TS) is a neurocutaneous genetically inherited disease with variable penetrance characterized by dysplasias and hamartomas affecting multiple organs. MR is the imaging method of choice to demonstrate structural brain lesions in TS. To compare MR sequences and determine which is most useful for the demonstration of each type of brain lesion in TS patients. We reviewed MR scans of 18 TS patients for the presence of cortical tubers, white matter lesions (radial bands), subependymal nodules, and subependymal giant cell astrocytoma (SGCA) on the following sequences: (1) T1-weighted spin-echo (T1 SE) images before and after gadolinium (Gd) injection; (2) nonenhanced T1 SE sequence with an additional magnetization transfer contrast medium pulse on resonance (T1 SE/MTC); and (3) fluid-attenuated inversion recovery (FLAIR) sequence. Cortical tubers were found in significantly (P<0.05) larger numbers and more conspicuously in FLAIR and T1 SE/MTC sequences. The T1 SE/MTC sequence was far superior to other methods in detecting white matter lesions (P<0.01). There was no significant difference between the T1 SE/MTC and T1 SE (before and after Gd injection) sequences in the detection of subependymal nodules; FLAIR sequence showed less sensitivity than the others in identifying the nodules. T1 SE sequences after Gd injection demonstrated better the limits of the SGCA. We demonstrated the importance of appropriate MRI sequences for diagnosis of the most frequent brain lesions in TS. Our study reinforces the fact that each sequence has a particular application according to the type of TS lesion. Gd injection might be useful in detecting SGCA; however, the parameters of size and location are also important for a presumptive diagnosis of these tumors. (orig.)

  14. Development of a Novel Fluorescence Assay Based on the Use of the Thrombin-Binding Aptamer for the Detection of O6-Alkylguanine-DNA Alkyltransferase Activity

    Directory of Open Access Journals (Sweden)

    Maria Tintoré

    2010-01-01

    Full Text Available Human O6-alkylguanine-DNA alkyltransferase (hAGT is a DNA repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O6 position of guanine. Here, we developed a real-time fluorescence hAGT activity assay that is based on the detection of conformational changes of the thrombin-binding aptamer (TBA. The quadruplex structure of TBA is disrupted when a central guanine is replaced by an O6-methyl-guanine. The sequence also contains a fluorophore (fluorescein and a quencher (dabsyl attached to the opposite ends. In the unfolded structure, the fluorophore and the quencher are separated. When hAGT removes the methyl group from the central guanine of TBA, it folds back immediately into its quadruplex structure. Consequently, the fluorophore and the quencher come into close proximity, thereby resulting in decreased fluorescence intensity. Here, we developed a new method to quantify the hAGT without using radioactivity. This new fluorescence resonance energy transfer assay has been designed to detect the conformational change of TBA that is induced by the removal of the O6-methyl group.

  15. CATH: an expanded resource to predict protein function through structure and sequence

    Science.gov (United States)

    Dawson, Natalie L.; Lewis, Tony E.; Das, Sayoni; Lees, Jonathan G.; Lee, David; Ashford, Paul; Orengo, Christine A.; Sillitoe, Ian

    2017-01-01

    The latest version of the CATH-Gene3D protein structure classification database has recently been released (version 4.1, http://www.cathdb.info). The resource comprises over 300 000 domain structures and over 53 million protein domains classified into 2737 homologous superfamilies, doubling the number of predicted protein domains in the previous version. The daily-updated CATH-B, which contains our very latest domain assignment data, provides putative classifications for over 100 000 additional protein domains. This article describes developments to the CATH-Gene3D resource over the last two years since the publication in 2015, including: significant increases to our structural and sequence coverage; expansion of the functional families in CATH; building a support vector machine (SVM) to automatically assign domains to superfamilies; improved search facilities to return alignments of query sequences against multiple sequence alignments; the redesign of the web pages and download site. PMID:27899584

  16. Intermediate divergence levels maximize the strength of structure-sequence correlations in enzymes and viral proteins.

    Science.gov (United States)

    Jackson, Eleisha L; Shahmoradi, Amir; Spielman, Stephanie J; Jack, Benjamin R; Wilke, Claus O

    2016-07-01

    Structural properties such as solvent accessibility and contact number predict site-specific sequence variability in many proteins. However, the strength and significance of these structure-sequence relationships vary widely among different proteins, with absolute correlation strengths ranging from 0 to 0.8. In particular, two recent works have made contradictory observations. Yeh et al. (Mol. Biol. Evol. 31:135-139, 2014) found that both relative solvent accessibility (RSA) and weighted contact number (WCN) are good predictors of sitewise evolutionary rate in enzymes, with WCN clearly out-performing RSA. Shahmoradi et al. (J. Mol. Evol. 79:130-142, 2014) considered these same predictors (as well as others) in viral proteins and found much weaker correlations and no clear advantage of WCN over RSA. Because these two studies had substantial methodological differences, however, a direct comparison of their results is not possible. Here, we reanalyze the datasets of the two studies with one uniform analysis pipeline, and we find that many apparent discrepancies between the two analyses can be attributed to the extent of sequence divergence in individual alignments. Specifically, the alignments of the enzyme dataset are much more diverged than those of the virus dataset, and proteins with higher divergence exhibit, on average, stronger structure-sequence correlations. However, the highest structure-sequence correlations are observed at intermediate divergence levels, where both highly conserved and highly variable sites are present in the same alignment.

  17. Integrating sequencing technologies in personal genomics: optimal low cost reconstruction of structural variants.

    Directory of Open Access Journals (Sweden)

    Jiang Du

    2009-07-01

    Full Text Available The goal of human genome re-sequencing is obtaining an accurate assembly of an individual's genome. Recently, there has been great excitement in the development of many technologies for this (e.g. medium and short read sequencing from companies such as 454 and SOLiD, and high-density oligo-arrays from Affymetrix and NimbelGen, with even more expected to appear. The costs and sensitivities of these technologies differ considerably from each other. As an important goal of personal genomics is to reduce the cost of re-sequencing to an affordable point, it is worthwhile to consider optimally integrating technologies. Here, we build a simulation toolbox that will help us optimally combine different technologies for genome re-sequencing, especially in reconstructing large structural variants (SVs. SV reconstruction is considered the most challenging step in human genome re-sequencing. (It is sometimes even harder than de novo assembly of small genomes because of the duplications and repetitive sequences in the human genome. To this end, we formulate canonical problems that are representative of issues in reconstruction and are of small enough scale to be computationally tractable and simulatable. Using semi-realistic simulations, we show how we can combine different technologies to optimally solve the assembly at low cost. With mapability maps, our simulations efficiently handle the inhomogeneous repeat-containing structure of the human genome and the computational complexity of practical assembly algorithms. They quantitatively show how combining different read lengths is more cost-effective than using one length, how an optimal mixed sequencing strategy for reconstructing large novel SVs usually also gives accurate detection of SNPs/indels, how paired-end reads can improve reconstruction efficiency, and how adding in arrays is more efficient than just sequencing for disentangling some complex SVs. Our strategy should facilitate the sequencing of

  18. Computer-Aided Design of RNA Origami Structures.

    Science.gov (United States)

    Sparvath, Steffen L; Geary, Cody W; Andersen, Ebbe S

    2017-01-01

    RNA nanostructures can be used as scaffolds to organize, combine, and control molecular functionalities, with great potential for applications in nanomedicine and synthetic biology. The single-stranded RNA origami method allows RNA nanostructures to be folded as they are transcribed by the RNA polymerase. RNA origami structures provide a stable framework that can be decorated with functional RNA elements such as riboswitches, ribozymes, interaction sites, and aptamers for binding small molecules or protein targets. The rich library of RNA structural and functional elements combined with the possibility to attach proteins through aptamer-based binding creates virtually limitless possibilities for constructing advanced RNA-based nanodevices.In this chapter we provide a detailed protocol for the single-stranded RNA origami design method using a simple 2-helix tall structure as an example. The first step involves 3D modeling of a double-crossover between two RNA double helices, followed by decoration with tertiary motifs. The second step deals with the construction of a 2D blueprint describing the secondary structure and sequence constraints that serves as the input for computer programs. In the third step, computer programs are used to design RNA sequences that are compatible with the structure, and the resulting outputs are evaluated and converted into DNA sequences to order.

  19. Sequence determination and modeling of structural motifs for the smallest monomeric aminoacyl-tRNA synthetase.

    OpenAIRE

    Hou, Y M; Shiba, K; Mottes, C; Schimmel, P.

    1991-01-01

    Polypeptide chains of 19 previously studied Escherichia coli aminoacyl-tRNA synthetases are as large as 951 amino acids and, depending on the enzyme, have quaternary structures of alpha, alpha 2, alpha 2 beta 2, and alpha 4. These enzymes have been organized into two classes which are defined by sequence motifs that are associated with specific three-dimensional structures. We isolated, cloned, and sequenced the previously uncharacterized gene for E. coli cysteine-tRNA synthetase (EC 6.1.1.16...

  20. Multi-scaling hierarchical structure analysis on the sequence of E. coli complete genome

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    We have applied the newly developed hierarchical structure theory for complex systems to analyze the multi-scaling structures of the nucleotide density distribution along a linear DNA sequence from the complete Escherichia coli genome. The hierarchical symmetry in the nucleotide density distribution was demonstrated. In particular, we have shown that the G, C density distribution that represents a strong H-bonding between the two DNA chains is more coherent with smaller similarity parameter compared to that of A, T density distribution, indicating a better organized multi-scaling fluctuation field for G, C density distribution along the genome sequence. The biological significance of these findings is under investigation.

  1. Circular dichroism spectroscopic studies on structures formed by telomeric DNA sequences in vitro

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Telomere plays an important role in cellular processes, such as cell aging, death and carcinogenisis. Having special sequences, it can form quadruplex structure in vitro. Circular dichroism (CD) spectroscopic studies show that TTAGGG, (TTAGGG)2 and (TTAGGG)4 can all form quadruplex in vitro and exist mainly as parallel quadruplex without metal ions. Both K+ and Na+ can stabilize the tetrameric structure and facilitate the forming of anti-parallel conformation. Furthermore, the conformations of quadruplex can also be affected by sequence length, the nature and concentration of metal ions.

  2. The structure contours of the Calico sequence boundary in the Kaiparowits Plateau, southern Utah (csbstrc*g)

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — This is a polygon coverage of the structure contours of the Calico sequence boundary in the Kaiparowits Plateau, southern Utah. Sequence boundary elevations are...

  3. Typhoidal Salmonellae: Use of Multi-Locus Sequence Typing to Determine Population Structure.

    Science.gov (United States)

    Sharma, Priyanka; Dahiya, Sushila; Balaji, Veeraraghavan; Kanga, Anil; Panda, Preetilata; Das, Rashna; Dhanraju, Anbumani; Mendiratta, Deepak Kumar; Sood, Seema; Das, Bimal Kumar; Kapil, Arti

    2016-01-01

    Enteric fever is an invasive infection predominantly caused by Salmonella enterica serovars Typhi and Paratyphi A. The pathogens have evolved from other nontyphoidal salmonellaeto become invasive and host restricted. Emergence of antimicrobial resistance in typhoidal salmonellae in some countries is a major therapeutic concern as the travelers returning from endemic countries carry resistant strains to non endemic areas. In order to understand the epidemiology and to design disease control strategies molecular typing of the pathogen is very important. We performed Multilocus Sequence Typing (MLST) of 251 S. Typhi and 18 S. Paratyphi strains isolated from enteric fever patients from seven centers across India during 2010-2013to determine the population structure and prevalence of MLST sequence types in India. MLST analysis revealed the presence of five sequence types (STs) of typhoidal salmonellae in India namely ST1, ST2 and ST3 for S. Typhi and ST85 and ST129 for S. Paratyphi A.S. Typhi strains showed monophyletic lineage and clustered in to 3 Sequence Types-ST1, ST2 and ST3 and S. Paratyphi A isolates segregated in two sequence types ST85 and ST129 respectively. No association was found between antimicrobial susceptibility and sequence types. This study found ST1 as the most prevalent sequence type of S. Typhi in India followed by ST2, which is in concordance with previous studies and MLST database. In addition a rare sequence type ST3 has been found which is reported for the first time from the Indian subcontinent. Amongst S. Paratyphi A, the most common sequence type is ST129 as also reported from other parts of world. This distribution and prevalence suggest the common spread of the sequence types across the globe and these findings can help in understanding the disease distribution.

  4. Structure and chromosomal localization of DNA sequences related to ribosomal subrepeats in Vicia faba.

    Science.gov (United States)

    Maggini, F; Cremonini, R; Zolfino, C; Tucci, G F; D'Ovidio, R; Delre, V; DePace, C; Scarascia Mugnozza, G T; Cionini, P G

    1991-05-01

    Subrepeating sequences of 325 bp found in the ribosomal intergenic spacer (IGS) of Vicia faba and responsible for variations in the length of the polycistronic units for rRNA were isolated and used as probes for in situ hybridization. Hybridization occurs at many regions of the metaphase chromosomes besides those bearing rRNA genes, namely chromosome ends and all the heterochromatic regions revealed by enhanced fluorescence after quinacrine staining. The DNA homologous to the 325 bp repeats that does not reside in the IGS was isolated, cloned and sequenced. It is composed of tandemly arranged 336 bp elements, each comprising two highly related 168 bp sequences. This structure is very similar to that of the IGS repeats and ca. 75% nucleotide sequence identity can be observed between these and the 168 bp doublets. The most obvious difference lies in the deletion, in the former, of a 14 bp segment from one of the two related sequences. It is hypothesized that the IGS repeats are derived from the 336 bp elements and have been transposed to ribosomal cistrons from other genome fractions. The possible relations between these sequences and others with similar structural features found in other species are discussed.

  5. Using maximum entropy model to predict protein secondary structure with single sequence.

    Science.gov (United States)

    Ding, Yong-Sheng; Zhang, Tong-Liang; Gu, Quan; Zhao, Pei-Ying; Chou, Kuo-Chen

    2009-01-01

    Prediction of protein secondary structure is somewhat reminiscent of the efforts by many previous investigators but yet still worthy of revisiting it owing to its importance in protein science. Several studies indicate that the knowledge of protein structural classes can provide useful information towards the determination of protein secondary structure. Particularly, the performance of prediction algorithms developed recently have been improved rapidly by incorporating homologous multiple sequences alignment information. Unfortunately, this kind of information is not available for a significant amount of proteins. In view of this, it is necessary to develop the method based on the query protein sequence alone, the so-called single-sequence method. Here, we propose a novel single-sequence approach which is featured by that various kinds of contextual information are taken into account, and that a maximum entropy model classifier is used as the prediction engine. As a demonstration, cross-validation tests have been performed by the new method on datasets containing proteins from different structural classes, and the results thus obtained are quite promising, indicating that the new method may become an useful tool in protein science or at least play a complementary role to the existing protein secondary structure prediction methods.

  6. Sequence and structural analyses of nuclear export signals in the NESdb database.

    Science.gov (United States)

    Xu, Darui; Farmer, Alicia; Collett, Garen; Grishin, Nick V; Chook, Yuh Min

    2012-09-01

    We compiled >200 nuclear export signal (NES)-containing CRM1 cargoes in a database named NESdb. We analyzed the sequences and three-dimensional structures of natural, experimentally identified NESs and of false-positive NESs that were generated from the database in order to identify properties that might distinguish the two groups of sequences. Analyses of amino acid frequencies, sequence logos, and agreement with existing NES consensus sequences revealed strong preferences for the Φ1-X(3)-Φ2-X(2)-Φ3-X-Φ4 pattern and for negatively charged amino acids in the nonhydrophobic positions of experimentally identified NESs but not of false positives. Strong preferences against certain hydrophobic amino acids in the hydrophobic positions were also revealed. These findings led to a new and more precise NES consensus. More important, three-dimensional structures are now available for 68 NESs within 56 different cargo proteins. Analyses of these structures showed that experimentally identified NESs are more likely than the false positives to adopt α-helical conformations that transition to loops at their C-termini and more likely to be surface accessible within their protein domains or be present in disordered or unobserved parts of the structures. Such distinguishing features for real NESs might be useful in future NES prediction efforts. Finally, we also tested CRM1-binding of 40 NESs that were found in the 56 structures. We found that 16 of the NES peptides did not bind CRM1, hence illustrating how NESs are easily misidentified.

  7. Prediction of Spontaneous Protein Deamidation from Sequence-Derived Secondary Structure and Intrinsic Disorder.

    Directory of Open Access Journals (Sweden)

    J Ramiro Lorenzo

    Full Text Available Asparagine residues in proteins undergo spontaneous deamidation, a post-translational modification that may act as a molecular clock for the regulation of protein function and turnover. Asparagine deamidation is modulated by protein local sequence, secondary structure and hydrogen bonding. We present NGOME, an algorithm able to predict non-enzymatic deamidation of internal asparagine residues in proteins in the absence of structural data, using sequence-based predictions of secondary structure and intrinsic disorder. Compared to previous algorithms, NGOME does not require three-dimensional structures yet yields better predictions than available sequence-only methods. Four case studies of specific proteins show how NGOME may help the user identify deamidation-prone asparagine residues, often related to protein gain of function, protein degradation or protein misfolding in pathological processes. A fifth case study applies NGOME at a proteomic scale and unveils a correlation between asparagine deamidation and protein degradation in yeast. NGOME is freely available as a webserver at the National EMBnet node Argentina, URL: http://www.embnet.qb.fcen.uba.ar/ in the subpage "Protein and nucleic acid structure and sequence analysis".

  8. Facile Discovery of Cell-Surface Protein Targets of Cancer Cell Aptamers.

    Science.gov (United States)

    Bing, Tao; Shangguan, Dihua; Wang, Yinsheng

    2015-10-01

    Cancer biomarker discovery constitutes a frontier in cancer research. In recent years, cell-binding aptamers have become useful molecular probes for biomarker discovery. However, there are few successful examples, and the critical barrier resides in the identification of the cell-surface protein targets for the aptamers, where only a limited number of aptamer targets have been identified so far. Herein, we developed a universal SILAC-based quantitative proteomic method for target discovery of cell-binding aptamers. The method allowed for distinguishing specific aptamer-binding proteins from nonspecific proteins based on abundance ratios of proteins bound to aptamer-carrying bait and control bait. In addition, we employed fluorescently labeled aptamers for monitoring and optimizing the binding conditions. We were able to identify and validate selectin L and integrin α4 as the protein targets for two previously reported aptamers, Sgc-3b and Sgc-4e, respectively. This strategy should be generally applicable for the discovery of protein targets for other cell-binding aptamers, which will promote the applications of these aptamers.

  9. RNA aptamers inhibit the growth of the fish pathogen viral hemorrhagic septicemia virus (VHSV).

    Science.gov (United States)

    Punnarak, Porntep; Santos, Mudjekeewis D; Hwang, Seong Don; Kondo, Hidehiro; Hirono, Ikuo; Kikuchi, Yo; Aoki, Takashi

    2012-12-01

    Viral hemorrhagic septicemia virus (VHSV) is a serious disease impacting wild and cultured fish worldwide. Hence, an effective therapeutic method against VHSV infection needs to be developed. Aptamer technology is a new and promising method for diagnostics and therapeutics. It revolves around the use of an aptamer molecule, an artificial ligand (nucleic acid or protein), which has the capacity to recognize target molecules with high affinity and specificity. Here, we aimed at selecting RNA aptamers that can specifically bind to and inhibit the growth of a strain of fish VHSV both in vitro and in vivo. Three VHSV-specific RNA aptamers (F1, F2, and C6) were selected from a pool of artificially and randomly produced oligonucleotides using systematic evolution of ligands by exponential enrichment. The three RNA aptamers showed obvious binding to VHSV in an electrophoretic mobility shift assay but not to other tested viruses. The RNA aptamers were tested for their ability to inhibit VHSV in vitro using hirame natural embryo (HINAE) cells. Cytopathic effect and plaque assays showed that all aptamers inhibited the growth of VHSV in HINAE cells. In vivo tests using RNA aptamers produced by Rhodovulum sulfidophilum showed that extracellular RNA aptamers inhibited VHSV infection in Japanese flounder. These results suggest that the RNA aptamers are a useful tool for protection against VHSV infection in Japanese flounder.

  10. Modified AS1411 Aptamer Suppresses Hepatocellular Carcinoma by Up-Regulating Galectin-14.

    Science.gov (United States)

    Cho, Yuri; Lee, Yun Bin; Lee, Jeong-Hoon; Lee, Dong Hyeon; Cho, Eun Ju; Yu, Su Jong; Kim, Yoon Jun; Kim, Jong In; Im, Jong Hun; Lee, Jung Hwan; Oh, Eun Ju; Yoon, Jung-Hwan

    2016-01-01

    Aptamers are small synthetic oligonucleotides that bind to target proteins with high specificity and affinity. AS1411 is an aptamer that binds to nucleolin, which is overexpressed in the cytoplasm and occurs on the surface of cancer cells. We investigated the therapeutic potential of aptamers in hepatocellular carcinoma (HCC) by evaluating anti-tumor effects and confirming the affinity and specificity of AS1411- and modified AS1411-aptamers in HCC cells. Cell growth was assessed using the MTS assay, and cell death signaling was explored by immunoblot analysis. Fluorescence-activated cell sorting was performed to evaluate the affinity and specificity of AS1411-aptamers in SNU-761 HCC cells. We investigated the in vivo effects of the AS1411-aptamer using BALB/c nude mice in a subcutaneous xenograft model with SNU-761 cells. Treatment with a modified AS1411-aptamer significantly decreased in vitro (under normoxic [P = 0.035] and hypoxic [P = 0.018] conditions) and in vivo (under normoxic conditions, P = 0.041) HCC cell proliferation compared to control aptamers. AS1411- and control aptamers failed to control HCC cell proliferation. However, AS1411- and the modified AS1411-aptamer did not induce caspase activation. Decrease in cell growth by AS1411 or modified AS1411 was not prevented by caspase or necrosis inhibitors. In a microarray, AS1411 significantly enhanced galectin-14 expression. Suppression of HCC cell proliferation by the modified AS1411-aptamer was attenuated by galectin-14 siRNA transfection. Modified AS1411-aptamer suppressed HCC cell growth in vitro and in vivo by up-regulating galectin-14 expressions. Modified AS1411-aptamers may have therapeutic potential as a novel targeted therapy for HCC.

  11. [Characterization of 5S rRNA gene sequence and secondary structure in gymnosperms].

    Science.gov (United States)

    Liu, Zhan-Lin; Zhang, Da-Ming; Wang, Xiao-Ru

    2003-01-01

    In higher plants the primary and the secondary structures of 5S ribosomal RNA gene are considered highly conservative. Little is known about the 5S rRNA gene structure, organization and variation in gyimnosperms. In this study we analyzed sequence and structure variation of 5S rRNA gene in Pinus through cloning and sequencing multiple copies of 5S rDNA repeats from individual trees of five pines, P. bungeana, P. tabulaeformis, P. yunnanensis, P. massoniana and P. densata. Pinus bungeana is from the subgenus Strobus while the other four are from the subgenus Pinus (diploxylon pines). Our results revealed variations in both primary and secondary structure among copies of 5S rDNA within individual genomes and between species. 5S rRNA gene in Pinus is 120 bp long in most of the 122 clones we sequenced except for one or two deletions in three clones. Among these clones 50 unique sequences were identified and they were shared by different pine species. Our sequences were compared to 13 sequences each representing a different gymnosperm species, and to six sequences representing both angiosperm monocots and dicots. Average sequence similarity was 97.1% among Pinus species and 94.3% between Pinus and other gymnosperms. Between gymnosperms and angiosperms the sequence similarity decreased to 88.1%. Similar to other molecular data, significant sequence divergence was found between the two Pinus subgenera. The 5S gene tree (neighbor-joining tree) grouped the four diploxylon pines together and separated them distinctly from P. bungeana. Comparison of sequence divergence within individuals and between species suggested that concerted evolution has been very weak especially after the divergence of the four diploxylon pines. The phylogenetic information contained in the 5S rRNA gene is limited due to its shorter length and the difficulties in identifying orthologous and paralogous copies of rDNA multigene family further complicate its phylogenetic application. Pinus densata is a

  12. Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes.

    Science.gov (United States)

    MacGregor, E A; Janecek, S; Svensson, B

    2001-03-09

    The hydrolases and transferases that constitute the alpha-amylase family are multidomain proteins, but each has a catalytic domain in the form of a (beta/alpha)(8)-barrel, with the active site being at the C-terminal end of the barrel beta-strands. Although the enzymes are believed to share the same catalytic acids and a common mechanism of action, they have been assigned to three separate families - 13, 70 and 77 - in the classification scheme for glycoside hydrolases and transferases that is based on amino acid sequence similarities. Each enzyme has one glutamic acid and two aspartic acid residues necessary for activity, while most enzymes of the family also contain two histidine residues critical for transition state stabilisation. These five residues occur in four short sequences conserved throughout the family, and within such sequences some key amino acid residues are related to enzyme specificity. A table is given showing motifs distinctive for each specificity as extracted from 316 sequences, which should aid in identifying the enzyme from primary structure information. Where appropriate, existing problems with identification of some enzymes of the family are pointed out. For enzymes of known three-dimensional structure, action is discussed in terms of molecular architecture. The sequence-specificity and structure-specificity relationships described may provide useful pointers for rational protein engineering.

  13. R3D-2-MSA: the RNA 3D structure-to-multiple sequence alignment server.

    Science.gov (United States)

    Cannone, Jamie J; Sweeney, Blake A; Petrov, Anton I; Gutell, Robin R; Zirbel, Craig L; Leontis, Neocles

    2015-07-01

    The RNA 3D Structure-to-Multiple Sequence Alignment Server (R3D-2-MSA) is a new web service that seamlessly links RNA three-dimensional (3D) structures to high-quality RNA multiple sequence alignments (MSAs) from diverse biological sources. In this first release, R3D-2-MSA provides manual and programmatic access to curated, representative ribosomal RNA sequence alignments from bacterial, archaeal, eukaryal and organellar ribosomes, using nucleotide numbers from representative atomic-resolution 3D structures. A web-based front end is available for manual entry and an Application Program Interface for programmatic access. Users can specify up to five ranges of nucleotides and 50 nucleotide positions per range. The R3D-2-MSA server maps these ranges to the appropriate columns of the corresponding MSA and returns the contents of the columns, either for display in a web browser or in JSON format for subsequent programmatic use. The browser output page provides a 3D interactive display of the query, a full list of sequence variants with taxonomic information and a statistical summary of distinct sequence variants found. The output can be filtered and sorted in the browser. Previous user queries can be viewed at any time by resubmitting the output URL, which encodes the search and re-generates the results. The service is freely available with no login requirement at http://rna.bgsu.edu/r3d-2-msa.

  14. Duplex Identification of Staphylococcus aureus by Aptamer and Gold Nanoparticles.

    Science.gov (United States)

    Chang, Tianjun; Wang, Libo; Zhao, Kexu; Ge, Yu; He, Meng; Li, Gang

    2016-06-01

    Staphylococcus aureus is the top common pathogen causing infections and food poisoning. Identification of S. aureus is crucial for the disease diagnosis and regulation of food hygiene. Herein, we report an aptamer-AuNPs based method for duplex identification of S. aureus. Using AuNPs as an indicator, SA23, an aptamer against S. aureus, can well identify its target from Escherichia coli, Listeria monocytogenes and Pseudomonas aeruginosa. Furthermore, we find citrate-coated AuNPs can strongly bind to S. aureus, but not bind to Salmonella enterica and Proteus mirabilis, which leads to different color changes in salt solution. This colorimetric response is capable of distinguishing S. aureus from S. enteritidis and P. mirabilis. Thus, using the aptasensor and AuNPs together, S. aureus can be accurately identified from the common pathogens. This duplex identification system is a promising platform for simple visual identification of S. aureus. Additionally, in the aptasensing process, bacteria are incubated with aptamers and then be removed before the aptamers adding to AuNPs, which may avoid the interactions between bacteria and AuNPs. This strategy can be potentially applied in principle to detect other cells by AuNPs-based aptasensors.

  15. Aptamer conjugated silver nanoparticles for the detection of interleukin 6

    Science.gov (United States)

    Locke, Andrea K.; Norwood, Nicole; Marks, Haley L.; Schechinger, Monika; Jackson, George W.; Graham, Duncan; Coté, Gerard L.

    2016-03-01

    The controlled assembly of plasmonic nanoparticles by a molecular binding event has emerged as a simple yet sensitive methodology for protein detection. Metallic nanoparticles (NPs) coated with functionalized aptamers can be utilized as biosensors by monitoring changes in particle optical properties, such as the LSPR shift and enhancement of the SERS spectra, in the presence of a target protein. Herein we test this method using two modified aptamers selected for the protein biomarker interleukin 6, an indicator of the dengue fever virus and other diseases including certain types of cancers, diabetes, and even arthritis. IL6 works by inducing an immunological response within the body that can be either anti-inflammatory or pro-inflammatory. The results show that the average hydrodynamic diameter of the NPs as measured by Dynamic Light Scattering was ~42 nm. After conjugation of the aptamers, the peak absorbance of the AgNPs shifted from 404 to 408 nm indicating a surface modification of the NPs due to the presence of the aptamer. Lastly, preliminary results were obtained showing an increase in SERS intensity occurs when the IL-6 protein was introduced to the conjugate solution but the assay will still need to be optimized in order for it to be able to monitor varying concentration changes within and across the desired range.

  16. Aptamer-based Field-Effect Biosensor for Tenofovir Detection

    Science.gov (United States)

    Aliakbarinodehi, N.; Jolly, P.; Bhalla, N.; Miodek, A.; De Micheli, G.; Estrela, P.; Carrara, S.

    2017-01-01

    During medical treatment it is critical to maintain the circulatory concentration of drugs within their therapeutic range. A novel biosensor is presented in this work to address the lack of a reliable point-of-care drug monitoring system in the market. The biosensor incorporates high selectivity and sensitivity by integrating aptamers as the recognition element and field-effect transistors as the signal transducer. The drug tenofovir was used as a model small molecule. The biointerface of the sensor is a binary self-assembled monolayer of specific thiolated aptamer and 6-mercapto-1-hexanol (MCH), whose ratio was optimized by electrochemical impedance spectroscopy measurements to enhance the sensitivity towards the specific target. Surface plasmon resonance, performed under different buffer conditions, shows optimum specific and little non-specific binding in phosphate buffered saline. The dose-response behavior of the field-effect biosensor presents a linear range between 1 nM and 100 nM of tenofovir and a limit of detection of 1.2 nM. Two non-specific drugs and one non-specific aptamer, tested as stringent control candidates, caused negligible responses. The applications were successfully extended to the detection of the drug in human serum. As demonstrated by impedance measurements, the aptamer-based sensors can be used for real-time drug monitoring. PMID:28294122

  17. Regulation of photosensitisation processes by an RNA aptamer

    Science.gov (United States)

    Thoa, Tran Thi Thanh; Minagawa, Noriko; Aigaki, Toshiro; Ito, Yoshihiro; Uzawa, Takanori

    2017-02-01

    One of the most powerful attributes of proteins is their ability to bind to and modulate the chemistry of cofactors and prosthetic groups. Here, we demonstrated the ability of an artificial nucleic acid (an aptamer) to similarly control the functionality of a non-biological element. Specifically, we selected an RNA aptamer that binds tris(bipyridine) ruthenium (II), Ru(bpy)32+, an inorganic complex that has attracted intense interest due to its photoredox chemistry, including its ability to split water by visible light. We found that a newly discovered aptamer strongly and enantioselectively binds Λ-Ru(bpy)32+ (Kd = 65 nM) and, in doing so, selectively suppresses deactivation via energy transfer, thereby elongating the lifetime of its photo-excited state by four-fold. The ability of the aptamer to enhance this important aspect of Ru(bpy)32+ chemistry illustrates a broader point concerning the potential power of combining in vitro-created biomolecules with non-biological reactants to perform enhanced chemical reactions.

  18. SYNTHESIS AND CHARACTERIZATION OF AROMATIC LIQUID CRYSTALLINE COPOLYESTERS WITH REGULAR SEQUENCE STRUCTURE

    Institute of Scientific and Technical Information of China (English)

    LIN Qinghuang; WANG Huifen; LIU Deshan; ZHOU Qixiang

    1990-01-01

    Several novel aromatic liquid crystalline copolyesters with regular sequence structure were prepared by melt Schotten-Baumann polycondensation via complex monomer. Polarizing microscope with hot stage,thermal analysis and X-ray diffraction were used to investigate the structure and properties of the copolyesters . The effects of structural units, such as flexible spacer, noncolinear meta-linked phenylene unit, crankshaft unit, kink with flexible bridging unit and various substituted benzene rings on melting temperature of aromatic copolyesters were studied and discussed on the basis of crystalline structure of the polymers.

  19. Sequence, Stability, Structure of G-Quadruplexes and Their Drug Interactions

    Science.gov (United States)

    Chen, Yuwei; Yang, Danzhou

    2012-01-01

    Although DNA is most widely known to store and pass along genetic information, the discovery of G-quadruplex structures has illuminated a new role of DNA in biology. DNA G-quadruplexes are four-stranded globular nucleic acid secondary structures formed in specific G-rich sequences with biological significance, such as human telomeres and oncogene promoters. This review focuses on the unimolecular DNA G-quadruplexes, which can readily form in solution under physiological conditions and are considered to be most biologically relevant. Available structural data show a great conformational diversity of unimolecular G-quadruplexes, amenable to small molecule drug targeting. The relationship of sequence, structure, and stability of unimolecular DNA G-quadruplexes, as well as the recent progress on interactions with small molecule compounds and insights into rational design of G-quadruplex-interactive molecules, will be discussed. PMID:22956454

  20. Gleaning structural and functional information from correlations in protein multiple sequence alignments.

    Science.gov (United States)

    Neuwald, Andrew F

    2016-06-01

    The availability of vast amounts of protein sequence data facilitates detection of subtle statistical correlations due to imposed structural and functional constraints. Recent breakthroughs using Direct Coupling Analysis (DCA) and related approaches have tapped into correlations believed to be due to compensatory mutations. This has yielded some remarkable results, including substantially improved prediction of protein intra- and inter-domain 3D contacts, of membrane and globular protein structures, of substrate binding sites, and of protein conformational heterogeneity. A complementary approach is Bayesian Partitioning with Pattern Selection (BPPS), which partitions related proteins into hierarchically-arranged subgroups based on correlated residue patterns. These correlated patterns are presumably due to structural and functional constraints associated with evolutionary divergence rather than to compensatory mutations. Hence joint application of DCA- and BPPS-based approaches should help sort out the structural and functional constraints contributing to sequence correlations.

  1. Welding sequence effects on residual stress distribution in offshore wind monopile structures

    Directory of Open Access Journals (Sweden)

    Ali Mehmanparast

    2016-01-01

    Full Text Available Residual stresses are often inevitably introduced into the material during the fabrication processes, such as welding, and are known to have significant effects on the subsequent fatigue crack growth behavior of welded structures. In this paper, the importance of welding sequence on residual stress distribution in engineering components has been reviewed. In addition, the findings available in the literature have been used to provide an accurate interpretation of the fatigue crack growth data on specimens extracted from the welded plates employed in offshore wind monopile structures. The results have been discussed in terms of the role of welding sequence in damage inspection and structural integrity assessment of offshore renewable energy structures.

  2. Sequence and secondary structure of the mitochondrial 16S ribosomal RNA gene of Ixodes scapularis.

    Science.gov (United States)

    Krakowetz, Chantel N; Chilton, Neil B

    2015-02-01

    The complete DNA sequences and secondary structure of the mitochondrial (mt) 16S ribosomal (r) RNA gene were determined for six Ixodes scapularis adults. There were 44 variable nucleotide positions in the 1252 bp sequence alignment. Most (95%) nucleotide alterations did not affect the integrity of the secondary structure of the gene because they either occurred at unpaired positions or represented compensatory changes that maintained the base pairing in helices. A large proportion (75%) of the intraspecific variation in DNA sequence occurred within Domains I, II and VI of the 16S gene. Therefore, several regions within this gene may be highly informative for studies of the population genetics and phylogeography of I. scapularis, a major vector of pathogens of humans and domestic animals in North America.

  3. The Structure of a Bernoulli Process Variation of the Fibonacci Sequence

    CERN Document Server

    Benson, Brian A

    2007-01-01

    We consider the structure of a variation of the Fibonacci sequence which is determined by a Bernoulli process. The associated structure of all Bernoulli variations of the Fibonacci sequence can be represented by a directed binary tree, which we denote X, with vertex labels representing the specific state of the recurrence variation. Since X is a binary tree, we can consider the term of a sequence variation given by a finite traversal of X represented by a binary code t. We then prove that the traversal of X that is the reflection of the digits of t gives exactly the integer term corresponding to t. We consider how to further this result with the statement of an additional conjecture. Finally, we give connections to Fibonacci expansions, the Stern-Brocot tree, and we apply our methods to the Three Hat Problem as seen in ``Puzzle Corner'' of the ``Technology Review'' magazine.

  4. Structure of the fully modified left-handed cyclohexene nucleic acid sequence GTGTACAC.

    Science.gov (United States)

    Robeyns, Koen; Herdewijn, Piet; Van Meervelt, Luc

    2008-02-13

    CeNA oligonucleotides consist of a phosphorylated backbone where the deoxyribose sugars are replaced by cyclohexene moieties. The X-ray structure determination and analysis of a fully modified octamer sequence GTGTACAC, which is the first crystal structure of a carbocyclic-based nucleic acid, is presented. This particular sequence was built with left-handed building blocks and crystallizes as a left-handed double helix. The helix can be characterized as belonging to the (mirrored) A-type family. Crystallographic data were processed up to 1.53 A, and the octamer sequence crystallizes in the space group R32. The sugar puckering is found to adopt the 3H2 half-chair conformation which mimics the C3'-endo conformation of the ribose sugar. The double helices stack on top of each other to form continuous helices, and static disorder is observed due to this end-to-end stacking.

  5. STUDY ON THE SEQUENCE STRUCTURE OF BUTADIENE-STYRENE RUBBER BY 13C-NMR METHOD Ⅲ. QUANTITATIVE CHARACTERIZATION OF SEQUENCE STRUCTURE

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiaonong; HU Liping; YAN Baozhen; JIAO Shuke

    1990-01-01

    The quantitative description of the sequence structure of emulsion-processed SBR and solution-processed SBR (by lithium catalyst)was carried out based on their spectral data of 13C-NMR.The calculating formulae which could be used to obtain diad concentration from the peak intensities of carbon spectra, average block length, average number of block, and the microstructure composition of the molecular chain were derived. The quantitative result showed that on the molecular chain styrene unit had the tendency to attach to trans-1,4 butadiene unit. The calculated result of the microstructure was in good agreement with that obtained through IR measurement.

  6. Characterization of the paclitaxel loaded chitosan graft Pluronic F127 copolymer micelles conjugate with a DNA aptamer targeting HER-2 overexpressing breast cancer cells

    Science.gov (United States)

    Thach Nguyen, Kim; Nguyen, Thu Ha; Do, Dinh Ho; Huan Le, Quang

    2017-03-01

    In this work we report the isolation of DNA aptamer that is specifically bound to a HER-2 overexpressing SK-BR-3 human breast cancer cell line, using SELEX strategy. Paclitaxel (PTX) loaded chitosan graft Pluronic F127 copolymer micelles conjugate with a DNA aptamer was synthesized and its structure was confirmed by TEM image. This binary mixed system consisting of DNA aptamer modified Pluronic F127 and chitosan could enhance PTX loading capacity and increase micelle stability. Morphology images confirmed the existence of PTX micelles, with an average size of approximately 86.22 ± 1.45 nm diameters. Drug release profile showed that the PTX conjugate maintained a sustained PTX release. From in vitro cell experiment it was shown that 89%–93%, 50%–58%, 55%–62%, 24%–28% and 2%–7% of the SK-BR-3, NS-VN-67, LH-VN-48, HT-VN-26 and NV-VN-31, respectively, were dead after 6–48 h. These results demonstrated a novel DNA aptamer-micelle assembly for efficient detection and a system for the delivery of PTX targeting specific HER-2 overexpressing. We have also successfully cultivated cancer tissues of explants from Vietnamese patients on a type I collagen substrate. The NS-VN-67, LH-VN-48, HT-VN-26 and NV-VN-31cell lines were used as cellular model sources for the study of chemotherapy drug in cancer.

  7. Novel electrochemical dual-aptamer-based sandwich biosensor using molybdenum disulfide/carbon aerogel composites and Au nanoparticles for signal amplification.

    Science.gov (United States)

    Fang, Lin-Xia; Huang, Ke-Jing; Liu, Yang

    2015-09-15

    A new electrochemical aptamer biosensor for the platelet-derived growth factor BB (PDGF-BB) detection has been developed based on the signal amplification of MoS2/carbon aerogel composites (MoS2/CA) and sandwich assay. A facile hydrothermal route assisted by L-cysteine was applied to synthesize CA incorporated flower-like MoS2 with the large surface active sites and good conductivity. The electrochemical aptasensor was constructed by sandwiching the PDGF-BB between a glassy carbon electrode modified with thiol-terminated PDGF-BB aptamer-1 (Apt1)/gold nanoparticles (AuNPs)/MoS2/CA and the AuNPs with thiol-terminated PDGF-BB aptamer-2 (Apt2) and 6-ferrocenyl hexanethiol (Fc). Fc-AuNPs-Apt2 acted as tracer and AuNPs/MoS2/CA were utilized as the biosensor platform to immobilize a large amount of capture aptamers, owing to their layered structure and high surface-to-volume ratio. Based on the sandwich format, a dual signal amplification strategy had been successfully developed with a wide linear response in the range of 0.001-10nM and a limit of detection of 0.3 pM. The developed assay demonstrated good selectivity and high sensitivity, indicating potential applications in bioanalysis and biomedicine.

  8. Didactical Structures as an Outcome of Research on Teaching-Learning Sequences? Special Issue

    Science.gov (United States)

    Lijnse, Piet; Klaassen, Kees

    2004-01-01

    This paper describes 'didactical structures' as a possible outcome of research on teaching-learning sequences. Starting from an explicit didactical perspective, in this case a so-called problem-posing approach, the research emphasis lies on the didactical quality with which this particular perspective can be put into classroom practice in the…

  9. Community structure of arbuscular mycorrhizal fungi in undisturbed vegetation revealed by analyses of LSU rdna sequences

    DEFF Research Database (Denmark)

    Rosendahl, Søren; Holtgrewe-Stukenbrock, Eva

    2004-01-01

    Arbuscular mycorrhizal fungi (AMF) form a mutualistic symbiosis with plant roots and are found in most ecosystems. In this study the community structure of AMF in a clade of the genus Glomus was examined in undisturbed costal grassland using LSU rDNA sequences amplified from roots of Hieracium pi...

  10. WebScipio: An online tool for the determination of gene structures using protein sequences

    Directory of Open Access Journals (Sweden)

    Waack Stephan

    2008-09-01

    Full Text Available Abstract Background Obtaining the gene structure for a given protein encoding gene is an important step in many analyses. A software suited for this task should be readily accessible, accurate, easy to handle and should provide the user with a coherent representation of the most probable gene structure. It should be rigorous enough to optimise features on the level of single bases and at the same time flexible enough to allow for cross-species searches. Results WebScipio, a web interface to the Scipio software, allows a user to obtain the corresponding coding sequence structure of a here given a query protein sequence that belongs to an already assembled eukaryotic genome. The resulting gene structure is presented in various human readable formats like a schematic representation, and a detailed alignment of the query and the target sequence highlighting any discrepancies. WebScipio can also be used to identify and characterise the gene structures of homologs in related organisms. In addition, it offers a web service for integration with other programs. Conclusion WebScipio is a tool that allows users to get a high-quality gene structure prediction from a protein query. It offers more than 250 eukaryotic genomes that can be searched and produces predictions that are close to what can be achieved by manual annotation, for in-species and cross-species searches alike. WebScipio is freely accessible at http://www.webscipio.org.

  11. Parallel dynamics and evolution: Protein conformational fluctuations and assembly reflect evolutionary changes in sequence and structure.

    Science.gov (United States)

    Marsh, Joseph A; Teichmann, Sarah A

    2014-02-01

    Protein structure is dynamic: the intrinsic flexibility of polypeptides facilitates a range of conformational fluctuations, and individual protein chains can assemble into complexes. Proteins are also dynamic in evolution: significant variations in secondary, tertiary and quaternary structure can be observed among divergent members of a protein family. Recent work has highlighted intriguing similarities between these structural and evolutionary dynamics occurring at various levels. Here we review evidence showing how evolutionary changes in protein sequence and structure are often closely related to local protein flexibility and disorder, large-scale motions and quaternary structure assembly. We suggest that these correspondences can be largely explained by neutral evolution, while deviations between structural and evolutionary dynamics can provide valuable functional insights. Finally, we address future prospects for the field and practical applications that arise from a deeper understanding of the intimate relationship between protein structure, dynamics, function and evolution.

  12. Label-free fluorescence detection of melamine with a truncated aptamer.

    Science.gov (United States)

    Gu, Chunmei; Xiang, Yu; Guo, Hongli; Shi, Hanchang

    2016-07-21

    The 2008 Chinese milk scandal caused by the adulteration of melamine encouraged the public to pay attention to melamine detection in milk products and other food stuffs. To allow simple and rapid detection of melamine, we previously isolated an 88 nt melamine aptamer (called Rd29C33) using the structure-switching SELEX. However, this 88 nt oligonucleotide is costly to synthesize, and may also complicate the rational design of biosensors for melamine detection. To overcome this obstacle, we truncated Rd29C33 at several sites, and a 34 nt Rd29C33-T7 melamine aptamer was finally found to show comparable binding affinity and better selectivity to melamine compared to the original 88 nt Rd29C33. Furthermore, a label-free bioassay method for melamine detection was designed by using Rd29C33-T7 and thiazole orange (TO). The addition of melamine to a mixture of Rd29C33-T7 and TO caused the release of TO from Rd29C33-T7, resulting in a decrease of the fluorescence intensity of the solution. A detection limit of 0.12 μM for melamine was achieved using this label-free method. Good recovery ranging from 82.6% to 97.2% for melamine detection in whole milk samples suggested the promise of this bioassay method for application in monitoring melamine in real food stuffs.

  13. A sequence-based survey of the complex structural organization of tumor genomes

    Energy Technology Data Exchange (ETDEWEB)

    Collins, Colin; Raphael, Benjamin J.; Volik, Stanislav; Yu, Peng; Wu, Chunxiao; Huang, Guiqing; Linardopoulou, Elena V.; Trask, Barbara J.; Waldman, Frederic; Costello, Joseph; Pienta, Kenneth J.; Mills, Gordon B.; Bajsarowicz, Krystyna; Kobayashi, Yasuko; Sridharan, Shivaranjani; Paris, Pamela; Tao, Quanzhou; Aerni, Sarah J.; Brown, Raymond P.; Bashir, Ali; Gray, Joe W.; Cheng, Jan-Fang; de Jong, Pieter; Nefedov, Mikhail; Ried, Thomas; Padilla-Nash, Hesed M.; Collins, Colin C.

    2008-04-03

    The genomes of many epithelial tumors exhibit extensive chromosomal rearrangements. All classes of genome rearrangements can be identified using End Sequencing Profiling (ESP), which relies on paired-end sequencing of cloned tumor genomes. In this study, brain, breast, ovary and prostate tumors along with three breast cancer cell lines were surveyed with ESP yielding the largest available collection of sequence-ready tumor genome breakpoints and providing evidence that some rearrangements may be recurrent. Sequencing and fluorescence in situ hybridization (FISH) confirmed translocations and complex tumor genome structures that include coamplification and packaging of disparate genomic loci with associated molecular heterogeneity. Comparison of the tumor genomes suggests recurrent rearrangements. Some are likely to be novel structural polymorphisms, whereas others may be bona fide somatic rearrangements. A recurrent fusion transcript in breast tumors and a constitutional fusion transcript resulting from a segmental duplication were identified. Analysis of end sequences for single nucleotide polymorphisms (SNPs) revealed candidate somatic mutations and an elevated rate of novel SNPs in an ovarian tumor. These results suggest that the genomes of many epithelial tumors may be far more dynamic and complex than previously appreciated and that genomic fusions including fusion transcripts and proteins may be common, possibly yielding tumor-specific biomarkers and therapeutic targets.

  14. Galaxy Structure as a Driver of the Star Formation Sequence Slope and Scatter

    Science.gov (United States)

    Whitaker, Katherine E.; 3D-HST Collaboration

    2016-01-01

    It is well established that (1) star-forming galaxies follow a relation between their star formation rate (SFR) and stellar mass (M*), the "star formation sequence," and (2) the SFRs of galaxies correlate with their structure, where star-forming galaxies are less concentrated than quiescent galaxies at fixed mass. In this talk, we consider whether the scatter and slope of the star formation sequence is correlated with systematic variations in the Sérsic indices, n, of galaxies across the SFR-M* plane. Using a mass-complete sample of 23,848 galaxies at 0.5 3D-HST photometric catalogs, we find that the scatter of the star formation sequence is related in part to galaxy structure; the scatter due to variations in n at fixed mass for star-forming galaxies ranges from 0.14 ± 0.02 dex at z ˜ 2 to 0.30 ± 0.04 dex at z unity for disk-like galaxies, galaxies with n > 2 (implying more dominant bulges) have significantly lower SFR/M* than the main ridgeline of the star formation sequence. These results suggest that bulges in massive z ˜ 2 galaxies are actively building up, where the stars in the central concentration are relatively young. At z < 1, the presence of older bulges within star-forming galaxies lowers global SFR/M*, decreasing the slope and contributing significantly to the scatter of the star formation sequence.

  15. SoftSearch: integration of multiple sequence features to identify breakpoints of structural variations.

    Directory of Open Access Journals (Sweden)

    Steven N Hart

    Full Text Available BACKGROUND: Structural variation (SV represents a significant, yet poorly understood contribution to an individual's genetic makeup. Advanced next-generation sequencing technologies are widely used to discover such variations, but there is no single detection tool that is considered a community standard. In an attempt to fulfil this need, we developed an algorithm, SoftSearch, for discovering structural variant breakpoints in Illumina paired-end next-generation sequencing data. SoftSearch combines multiple strategies for detecting SV including split-read, discordant read-pair, and unmated pairs. Co-localized split-reads and discordant read pairs are used to refine the breakpoints. RESULTS: We developed and validated SoftSearch using real and synthetic datasets. SoftSearch's key features are 1 not requiring secondary (or exhaustive primary alignment, 2 portability into established sequencing workflows, and 3 is applicable to any DNA-sequencing experiment (e.g. whole genome, exome, custom capture, etc.. SoftSearch identifies breakpoints from a small number of soft-clipped bases from split reads and a few discordant read-pairs which on their own would not be sufficient to make an SV call. CONCLUSIONS: We show that SoftSearch can identify more true SVs by combining multiple sequence features. SoftSearch was able to call clinically relevant SVs in the BRCA2 gene not reported by other tools while offering significantly improved overall performance.

  16. The nucleotide sequence and genome structure of mung bean yellow mosaic geminivirus.

    Science.gov (United States)

    Morinaga, T; Ikegami, M; Miura, K

    1993-01-01

    Complete nucleotide sequences of the infectious cloned DNA components (DNA 1 and DNA 2) of mung bean yellow mosaic virus (MYMV) were determined. MYMV DNA 1 and DNA 2 consists of 2,723 and 2,675 nucleotides respectively. DNA 1 and DNA 2 have little sequence similarity except for a region of approximately 200 bases which is almost identical in the two molecules. Analysis of open reading frames revealed nine potential coding regions for proteins of mol. wt. > 10,000, six in DNA 1 and three in DNA 2. The nucleotide sequence of MYMV DNA was compared with that of bean golden mosaic virus (BGMV), tomato golden mosaic virus (TGMV) and African cassava mosaic virus (ACMV). The 200-base region common to the two DNAs of each virus had little sequence similarity, except for a highly conserved 33-36 base sequence potentially capable of forming a stable hairpin structure. The potential coding regions in the MYMV DNAs had counterparts in the BGMV, TGMV and ACMV, suggesting an overall similarity in genome organization, except for absence of 1L3 in MYMV DNA 1. The most highly conserved ORFs, MYMV 1R1, BGMV 1R1, TGMV 1R1 and ACMV 1R1, are the putative genes for the coat proteins of MYMV, BGMV, TGMV and ACMV, respectively. MYMV 1L1 has also a high degree of sequence similarity with BGMV 1L1, TGMV 1L1 and ACMV 1L1.

  17. Structure-based identification of new high-affinity nucleosome binding sequences.

    Science.gov (United States)

    Battistini, Federica; Hunter, Christopher A; Moore, Irene K; Widom, Jonathan

    2012-06-29

    The substrate for the proteins that express genetic information in the cell is not naked DNA but an assembly of nucleosomes, where the DNA is wrapped around histone proteins. The organization of these nucleosomes on genomic DNA is influenced by the DNA sequence. Here, we present a structure-based computational approach that translates sequence information into the energy required to bend DNA into a nucleosome-bound conformation. The calculations establish the relationship between DNA sequence and histone octamer binding affinity. In silico selection using this model identified several new DNA sequences, which were experimentally found to have histone octamer affinities comparable to the highest-affinity sequences known. The results provide insights into the molecular mechanism through which DNA sequence information encodes its organization. A quantitative appreciation of the thermodynamics of nucleosome positioning and rearrangement will be one of the key factors in understanding the regulation of transcription and in the design of new promoter architectures for the purposes of tuning gene expression dynamics.

  18. Comparison of In-Solution Biorecognition Properties of Aptamers against Ochratoxin A

    Science.gov (United States)

    McKeague, Maureen; Velu, Ranganathan; De Girolamo, Annalisa; Valenzano, Stefania; Pascale, Michelangelo; Smith, McKenzie; DeRosa, Maria C.

    2016-01-01

    Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by several species of Aspergillus and Penicillium and frequently found as a natural contaminant in a wide range of food commodities. Novel and robust biorecognition agents for detecting this molecule are required. Aptamers are artificial nucleic acid ligands able to bind with high affinity and specificity to a given target molecule. In the last few years, three separate research groups have selected aptamers for ochratoxin A. While each of these three families of aptamers have been incorporated into various methods for detecting OTA, it is unclear if each aptamer candidate is better suited for a particular application. Here, we perform the first head-to-head comparison of solution-based binding parameters for these groups of aptamers. Based on our results, we provide recommendations for the appropriate choice of aptamer for incorporation into solution-based biorecognition assays and applications. PMID:27854269

  19. Role of sequence and structural polymorphism on the mechanical properties of amyloid fibrils.

    Directory of Open Access Journals (Sweden)

    Gwonchan Yoon

    Full Text Available Amyloid fibrils playing a critical role in disease expression, have recently been found to exhibit the excellent mechanical properties such as elastic modulus in the order of 10 GPa, which is comparable to that of other mechanical proteins such as microtubule, actin filament, and spider silk. These remarkable mechanical properties of amyloid fibrils are correlated with their functional role in disease expression. This suggests the importance in understanding how these excellent mechanical properties are originated through self-assembly process that may depend on the amino acid sequence. However, the sequence-structure-property relationship of amyloid fibrils has not been fully understood yet. In this work, we characterize the mechanical properties of human islet amyloid polypeptide (hIAPP fibrils with respect to their molecular structures as well as their amino acid sequence by using all-atom explicit water molecular dynamics (MD simulation. The simulation result suggests that the remarkable bending rigidity of amyloid fibrils can be achieved through a specific self-aggregation pattern such as antiparallel stacking of β strands (peptide chain. Moreover, we have shown that a single point mutation of hIAPP chain constituting a hIAPP fibril significantly affects the thermodynamic stability of hIAPP fibril formed by parallel stacking of peptide chain, and that a single point mutation results in a significant change in the bending rigidity of hIAPP fibrils formed by antiparallel stacking of β strands. This clearly elucidates the role of amino acid sequence on not only the equilibrium conformations of amyloid fibrils but also their mechanical properties. Our study sheds light on sequence-structure-property relationships of amyloid fibrils, which suggests that the mechanical properties of amyloid fibrils are encoded in their sequence-dependent molecular architecture.

  20. Training set reduction methods for protein secondary structure prediction in single-sequence condition.

    Science.gov (United States)

    Aydin, Zafer; Altunbasak, Yucel; Pakatci, Isa Kemal; Erdogan, Hakan

    2007-01-01

    Orphan proteins are characterized by the lack of significant sequence similarity to database proteins. To infer the functional properties of the orphans, more elaborate techniques that utilize structural information are required. In this regard, the protein structure prediction gains considerable importance. Secondary structure prediction algorithms designed for orphan proteins (also known as single-sequence algorithms) cannot utilize multiple alignments or alignment profiles, which are derived from similar proteins. This is a limiting factor for the prediction accuracy. One way to improve the performance of a single-sequence algorithm is to perform re-training. In this approach, first, the models used by the algorithm are trained by a representative set of proteins and a secondary structure prediction is computed. Then, using a distance measure, the original training set is refined by removing proteins that are dissimilar to the given protein. This step is followed by the re-estimation of the model parameters and the prediction of the secondary structure. In this paper, we compare training set reduction methods that are used to re-train the hidden semi-Markov models employed by the IPSSP algorithm [1]. We found that the composition based reduction method has the highest performance compared to the alignment based and the Chou-Fasman based reduction methods. In addition, threshold-based reduction performed better than the reduction technique that selects the first 80% of the dataset proteins.

  1. Predicting protein ligand binding sites by combining evolutionary sequence conservation and 3D structure.

    Directory of Open Access Journals (Sweden)

    John A Capra

    2009-12-01

    Full Text Available Identifying a protein's functional sites is an important step towards characterizing its molecular function. Numerous structure- and sequence-based methods have been developed for this problem. Here we introduce ConCavity, a small molecule binding site prediction algorithm that integrates evolutionary sequence conservation estimates with structure-based methods for identifying protein surface cavities. In large-scale testing on a diverse set of single- and multi-chain protein structures, we show that ConCavity substantially outperforms existing methods for identifying both 3D ligand binding pockets and individual ligand binding residues. As part of our testing, we perform one of the first direct comparisons of conservation-based and structure-based methods. We find that the two approaches provide largely complementary information, which can be combined to improve upon either approach alone. We also demonstrate that ConCavity has state-of-the-art performance in predicting catalytic sites and drug binding pockets. Overall, the algorithms and analysis presented here significantly improve our ability to identify ligand binding sites and further advance our understanding of the relationship between evolutionary sequence conservation and structural and functional attributes of proteins. Data, source code, and prediction visualizations are available on the ConCavity web site (http://compbio.cs.princeton.edu/concavity/.

  2. Predicting protein ligand binding sites by combining evolutionary sequence conservation and 3D structure.

    Science.gov (United States)

    Capra, John A; Laskowski, Roman A; Thornton, Janet M; Singh, Mona; Funkhouser, Thomas A

    2009-12-01

    Identifying a protein's functional sites is an important step towards characterizing its molecular function. Numerous structure- and sequence-based methods have been developed for this problem. Here we introduce ConCavity, a small molecule binding site prediction algorithm that integrates evolutionary sequence conservation estimates with structure-based methods for identifying protein surface cavities. In large-scale testing on a diverse set of single- and multi-chain protein structures, we show that ConCavity substantially outperforms existing methods for identifying both 3D ligand binding pockets and individual ligand binding residues. As part of our testing, we perform one of the first direct comparisons of conservation-based and structure-based methods. We find that the two approaches provide largely complementary information, which can be combined to improve upon either approach alone. We also demonstrate that ConCavity has state-of-the-art performance in predicting catalytic sites and drug binding pockets. Overall, the algorithms and analysis presented here significantly improve our ability to identify ligand binding sites and further advance our understanding of the relationship between evolutionary sequence conservation and structural and functional attributes of proteins. Data, source code, and prediction visualizations are available on the ConCavity web site (http://compbio.cs.princeton.edu/concavity/).

  3. Bayesian segmental models with multiple sequence alignment profiles for protein secondary structure and contact map prediction.

    Science.gov (United States)

    Chu, Wei; Ghahramani, Zoubin; Podtelezhnikov, Alexei; Wild, David L

    2006-01-01

    In this paper, we develop a segmental semi-Markov model (SSMM) for protein secondary structure prediction which incorporates multiple sequence alignment profiles with the purpose of improving the predictive performance. The segmental model is a generalization of the hidden Markov model where a hidden state generates segments of various length and secondary structure type. A novel parameterized model is proposed for the likelihood function that explicitly represents multiple sequence alignment profiles to capture the segmental conformation. Numerical results on benchmark data sets show that incorporating the profiles results in substantial improvements and the generalization performance is promising. By incorporating the information from long range interactions in beta-sheets, this model is also capable of carrying out inference on contact maps. This is an important advantage of probabilistic generative models over the traditional discriminative approach to protein secondary structure prediction. The Web server of our algorithm and supplementary materials are available at http://public.kgi.edu/-wild/bsm.html.

  4. Inferring the Clonal Structure of Viral Populations from Time Series Sequencing.

    Directory of Open Access Journals (Sweden)

    Donatien F Chedom

    2015-11-01

    Full Text Available RNA virus populations will undergo processes of mutation and selection resulting in a mixed population of viral particles. High throughput sequencing of a viral population subsequently contains a mixed signal of the underlying clones. We would like to identify the underlying evolutionary structures. We utilize two sources of information to attempt this; within segment linkage information, and mutation prevalence. We demonstrate that clone haplotypes, their prevalence, and maximum parsimony reticulate evolutionary structures can be identified, although the solutions may not be unique, even for complete sets of information. This is applied to a chain of influenza infection, where we infer evolutionary structures, including reassortment, and demonstrate some of the difficulties of interpretation that arise from deep sequencing due to artifacts such as template switching during PCR amplification.

  5. Selective Sequence for the Peptide-Triggered Phase Transition of Lyotropic Liquid-Crystalline Structures.

    Science.gov (United States)

    Liu, Qingtao; Dong, Yao-Da; Boyd, Ben J

    2016-05-24

    A novel concept of using mixed lipids to construct selective peptide-sequence-sensing lyotropic liquid-crystalline (LLC) dispersion systems was investigated. The LLC systems were constructed using a mixture of phytantriol, a lipid that forms lyotropic liquid-crystalline phases, and a novel synthesized peptide-lipid (peplipid) for sensing a target peptide with the RARAR sequence. The internal structure of the dispersed LLC particles was converted from the lamellar structure (liposomes) to the inverse bicontinuous cubic phase (cubosomes) in the presence of the target peptide. The addition of common human proteins did not induce any structural change, indicating a high selectivity of interaction with the target peptide. The concept has potential for the design of targeted controlled release drug delivery agents.

  6. Secondary structure, a missing component of sequence-based minimotif definitions.

    Directory of Open Access Journals (Sweden)

    David P Sargeant

    Full Text Available Minimotifs are short contiguous segments of proteins that have a known biological function. The hundreds of thousands of minimotifs discovered thus far are an important part of the theoretical understanding of the specificity of protein-protein interactions, posttranslational modifications, and signal transduction that occur in cells. However, a longstanding problem is that the different abstractions of the sequence definitions do not accurately capture the specificity, despite decades of effort by many labs. We present evidence that structure is an essential component of minimotif specificity, yet is not used in minimotif definitions. Our analysis of several known minimotifs as case studies, analysis of occurrences of minimotifs in structured and disordered regions of proteins, and review of the literature support a new model for minimotif definitions that includes sequence, structure, and function.

  7. Inferring Aftershock Sequence Properties and Tectonic Structure Using Empirical Signal Detectors

    Science.gov (United States)

    Junek, William N.; Kværna, Tormod; Pirli, Myrto; Schweitzer, Johannes; Harris, David B.; Dodge, Douglas A.; Woods, Mark T.

    2015-02-01

    Seismotectonic studies of the 2008 Storfjorden aftershock sequence were limited to data acquired by the permanent, but sparse, regional seismic network in the Svalbard archipelago. Storfjorden's remote location and harsh polar environment inhibited deployment of temporary seismometers that would have improved observations of sequence events. The lack of good station coverage prevented the detection and computation of hypocenter locations of many low magnitude events (mb < 2.5) in the NORSAR analyst-reviewed bulletin. As a result, the fine structure of the sequence's space-time distribution was not captured. In this study, an autonomous event detection and clustering framework is employed to build a more complete catalog of Storfjorden events using data from the Spitsbergen (SPITS) array. The new catalog allows the spatiotemporal distribution of seismicity within the fjord to be studied in greater detail. Information regarding the location of active event clusters provides a means of inferring the tectonic structure within the fault zone. The distribution of active clusters and moment tensor solutions for the Storfjorden sequence suggests there are at least two different structures within the fjord: a NE-SW trending linear feature with oblique-normal to strike-slip faulting and E-W trending normal faults.

  8. Evol and ProDy for bridging protein sequence evolution and structural dynamics

    Science.gov (United States)

    Mao, Wenzhi; Liu, Ying; Chennubhotla, Chakra; Lezon, Timothy R.; Bahar, Ivet

    2014-01-01

    Correlations between sequence evolution and structural dynamics are of utmost importance in understanding the molecular mechanisms of function and their evolution. We have integrated Evol, a new package for fast and efficient comparative analysis of evolutionary patterns and conformational dynamics, into ProDy, a computational toolbox designed for inferring protein dynamics from experimental and theoretical data. Using information-theoretic approaches, Evol coanalyzes conservation and coevolution profiles extracted from multiple sequence alignments of protein families with their inferred dynamics. Availability and implementation: ProDy and Evol are open-source and freely available under MIT License from http://prody.csb.pitt.edu/. Contact: bahar@pitt.edu PMID:24849577

  9. CMsearch: simultaneous exploration of protein sequence space and structure space improves not only protein homology detection but also protein structure prediction

    KAUST Repository

    Cui, Xuefeng

    2016-06-15

    Motivation: Protein homology detection, a fundamental problem in computational biology, is an indispensable step toward predicting protein structures and understanding protein functions. Despite the advances in recent decades on sequence alignment, threading and alignment-free methods, protein homology detection remains a challenging open problem. Recently, network methods that try to find transitive paths in the protein structure space demonstrate the importance of incorporating network information of the structure space. Yet, current methods merge the sequence space and the structure space into a single space, and thus introduce inconsistency in combining different sources of information. Method: We present a novel network-based protein homology detection method, CMsearch, based on cross-modal learning. Instead of exploring a single network built from the mixture of sequence and structure space information, CMsearch builds two separate networks to represent the sequence space and the structure space. It then learns sequence–structure correlation by simultaneously taking sequence information, structure information, sequence space information and structure space information into consideration. Results: We tested CMsearch on two challenging tasks, protein homology detection and protein structure prediction, by querying all 8332 PDB40 proteins. Our results demonstrate that CMsearch is insensitive to the similarity metrics used to define the sequence and the structure spaces. By using HMM–HMM alignment as the sequence similarity metric, CMsearch clearly outperforms state-of-the-art homology detection methods and the CASP-winning template-based protein structure prediction methods.

  10. Affinity Probe Capillary Electrophoresis Evaluation of Aptamer Binding to Campylobacter jejuni Bacteria

    Science.gov (United States)

    2009-11-01

    Affinity Probe Capillary Electrophoresis Evaluation of Aptamer Binding to Campylobacter jejuni Bacteria by Dimitra N. Stratis-Cullum, Sun...Aptamer Binding to Campylobacter jejuni Bacteria Dimitra N. Stratis-Cullum, Sun McMasters, and Paul M. Pellegrino Sensors and Electron Devices...To) 2007–2008 4. TITLE AND SUBTITLE Affinity Probe Capillary Electrophoresis Evaluation of Aptamer Binding to Campylobacter jejuni Bacteria 5a

  11. Trends in the Design and Development of Specific Aptamers Against Peptides and Proteins.

    Science.gov (United States)

    Tabarzad, Maryam; Jafari, Marzieh

    2016-04-01

    Aptamers are single stranded oligonucleotides, comparable to monoclonal antibodies (mAbs) in selectivity and affinity and have significant strategic properties in design, development and applications more than mAbs. Ease of design and development, simple chemical modification and the attachment of functional groups, easily handling and more adaptability with analytical methods, small size and adaptation with nanostructures are the valuable characteristics of aptamers in comparison to large protein based ligands. Among a broad range of targets that their specific aptamers developed, proteins and peptides have significant position according to the number of related studies performed so far. Since proteins control many of important physiological and pathological incidents in the living organisms, particularly human beings and because of the benefits of aptamers in clinical and analytical applications, aptamer related technologies in the field of proteins and peptides are under progress, exclusively. Currently, there is only one FDA approved therapeutic aptamer in the pharmaceutical market, which is specific to vascular endothelial growth factor and is prescribed for age related macular degenerative disease. Additionally, there are several aptamers in the different phases of clinical trials. Almost all of these aptamers are specific to clinically important peptide or protein targets. In addition, the application of protein specific aptamers in the design and development of targeted drug delivery systems and diagnostic biosensors is another interesting field of aptamer technology. In this review, significant efforts related to development and applications of aptamer technologies in proteins and peptides sciences were considered to emphasis on the importance of aptamers in medicinal and clinical applications.

  12. Stem-loop structures of the repetitive DNA sequences located at human centromeres

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, G.; Garcia, A.E.; Ratliff, R.; Moyzis, R.K. [Los Alamos National Lab., NM (United States); Catasti, P.; Hong, Lin; Yau, P. [California Univ., Davis, CA (United States). Dept. of Biological Chemistry; Bradbury, E.M. [Los Alamos National Lab., NM (United States)]|[California Univ., Davis, CA (United States). Dept. of Biological Chemistry

    1993-09-01

    The presence of the highly conserved repetitive DNA sequences in the human centromeres argues for a special role of these sequences in their biological functions - most likely achieved by the formation of unusual structures. This prompted us to carry out quantitative one- and two-dimensional nuclear magnetic resonance (lD/2D NMR) spectroscopy to determine the structural properties of the human centromeric repeats, d(AATGG){sub n.d}(CCATT){sub n}. The studies on centromeric DNAs reveal that the complementary sequence, d(AATGG){sub n.d}(CCATT){sub n}, adopts the usual Watson-Crick B-DNA duplex and the pyrimidine-rich d(CCATT){sub n} strand is essentially a random coil. However, the purine-rich d(AATGG){sub n} strand is shown to adopt unusual stem-loop structures for repeat lengths, n=2,3,4, and 6. In addition to normal Watson-Crick A{center_dot}T pairs, the stem-loop structures are stabilized by mismatch A{center_dot}G and G{center_dot}G pairs in the stem and G-G-A stacking in the loop. Stem-loop structures of d(AATGG)n are independently verified by gel electrophoresis and nuclease digestion studies. Thermal melting studies show that the DNA repeats, d(AATGG){sub n}, are as stable as the corresponding Watson-Crick duplex d(AATGG){sub n.d}(CCATT){sub n}. Therefore, the sequence d(AATGG){sub n} can, indeed, nucleate a stem-loop structure at little free-energy cost and if, during mitosis, they are located on the chromosome surface they can provide specific recognition sites for kinetochore function.

  13. How the Sequence of a Gene Specifies Structural Symmetry in Proteins.

    Directory of Open Access Journals (Sweden)

    Xiaojuan Shen

    Full Text Available Internal symmetry is commonly observed in the majority of fundamental protein folds. Meanwhile, sufficient evidence suggests that nascent polypeptide chains of proteins have the potential to start the co-translational folding process and this process allows mRNA to contain additional information on protein structure. In this paper, we study the relationship between gene sequences and protein structures from the viewpoint of symmetry to explore how gene sequences code for structural symmetry in proteins. We found that, for a set of two-fold symmetric proteins from left-handed beta-helix fold, intragenic symmetry always exists in their corresponding gene sequences. Meanwhile, codon usage bias and local mRNA structure might be involved in modulating translation speed for the formation of structural symmetry: a major decrease of local codon usage bias in the middle of the codon sequence can be identified as a common feature; and major or consecutive decreases in local mRNA folding energy near the boundaries of the symmetric substructures can also be observed. The results suggest that gene duplication and fusion may be an evolutionarily conserved process for this protein fold. In addition, the usage of rare codons and the formation of higher order of secondary structure near the boundaries of symmetric substructures might have coevolved as conserved mechanisms to slow down translation elongation and to facilitate effective folding of symmetric substructures. These findings provide valuable insights into our understanding of the mechanisms of translation and its evolution, as well as the design of proteins via symmetric modules.

  14. Auslander-Reiten sequences and $t$-structures on the homotopy category of an abelian category

    CERN Document Server

    Backelin, Erik

    2009-01-01

    Let $\\Cab$ be an abelian category and let $\\KC$ be the bounded homotopy category of cochain complexes in $\\Cab$. We consider a $t$-structure on $\\KC$ that maps to the standard $t$-structure on the derived category $\\DC$ under the localization functor. Let $\\A$ be the heart of the $t$-structure. In the case when $\\Cab$ has finite length we show that objects of $\\Cab$ correspond to projective objects of $\\A$ and that simple objects of $\\A$ (if they exist) are given by Auslander's and Reiten's almost split sequences in $\\Cab$.

  15. Linking experimental results, biological networks and sequence analysis methods using Ontologies and Generalised Data Structures.

    Science.gov (United States)

    Koehler, Jacob; Rawlings, Chris; Verrier, Paul; Mitchell, Rowan; Skusa, Andre; Ruegg, Alexander; Philippi, Stephan

    2005-01-01

    The structure of a closely integrated data warehouse is described that is designed to link different types and varying numbers of biological networks, sequence analysis methods and experimental results such as those coming from microarrays. The data schema is inspired by a combination of graph based methods and generalised data structures and makes use of ontologies and meta-data. The core idea is to consider and store biological networks as graphs, and to use generalised data structures (GDS) for the storage of further relevant information. This is possible because many biological networks can be stored as graphs: protein interactions, signal transduction networks, metabolic pathways, gene regulatory networks etc. Nodes in biological graphs represent entities such as promoters, proteins, genes and transcripts whereas the edges of such graphs specify how the nodes are related. The semantics of the nodes and edges are defined using ontologies of node and relation types. Besides generic attributes that most biological entities possess (name, attribute description), further information is stored using generalised data structures. By directly linking to underlying sequences (exons, introns, promoters, amino acid sequences) in a systematic way, close interoperability to sequence analysis methods can be achieved. This approach allows us to store, query and update a wide variety of biological information in a way that is semantically compact without requiring changes at the database schema level when new kinds of biological information is added. We describe how this datawarehouse is being implemented by extending the text-mining framework ONDEX to link, support and complement different bioinformatics applications and research activities such as microarray analysis, sequence analysis and modelling/simulation of biological systems. The system is developed under the GPL license and can be downloaded from http://sourceforge.net/projects/ondex/

  16. Aptamer-Functionalized Fluorescent Silica Nanoparticles for Highly Sensitive Detection of Leukemia Cells

    Science.gov (United States)

    Tan, Juntao; Yang, Nuo; Hu, Zixi; Su, Jing; Zhong, Jianhong; Yang, Yang; Yu, Yating; Zhu, Jianmeng; Xue, Dabin; Huang, Yingying; Lai, Zongqiang; Huang, Yong; Lu, Xiaoling; Zhao, Yongxiang

    2016-06-01

    A simple, highly sensitive method to detect leukemia cells has been developed based on aptamer-modified fluorescent silica nanoparticles (FSNPs). In this strategy, the amine-labeled Sgc8 aptamer was conjugated to carboxyl-modified FSNPs via amide coupling between amino and carboxyl groups. Sensitivity and specificity of Sgc8-FSNPs were assessed using flow cytometry and fluorescence microscopy. These results showed that Sgc8-FSNPs detected leukemia cells with high sensitivity and specificity. Aptamer-modified FSNPs hold promise for sensitive and specific detection of leukemia cells. Changing the aptamer may allow the FSNPs to detect other types of cancer cells.

  17. Development of a Quartz Crystal Microbalance Biosensor with Aptamers as Bio-recognition Element

    Directory of Open Access Journals (Sweden)

    Chunyan Yao

    2010-06-01

    Full Text Available The ultimate goal in any biosensor development project is its use for actual sample detection. Recently, there has been an interest in biosensors with aptamers as bio-recognition elements, but reported examples all deal with standards, not human serum. In order to verify the differences of aptamer-based biosensor and antibody-based biosensor in clinical detection, a comparison of the performance of aptamer-based and antibody-based quartz crystal microbalance (QCM biosensors for the detection of immunoglobulin E (IgE in human serum was carried out. Aptamers (or antibodies specific to IgE were immobilized on the gold surface of a quartz crystal. The frequency shifts of the QCM were measured. The linear range with the antibody (10–240 μg/L compared to that of the aptamer (2.5–200 μg/L, but a lower detection limit could be observed in the aptamer-based biosensor. The reproducibility of the two biosensors was comparable. The aptamers were equivalent or superior to antibodies in terms of specificity and sensitivity. In addition, the aptamer receptors could tolerate repeated affine layer regeneration after ligand binding and recycling of the biosensor with little loss of sensitivity. When stored for three weeks, the frequency shifts of the aptamer-coated crystals were all greater than 90% of those on the response at the first day.

  18. Structural and sequence analysis of imelysin-like proteins implicated in bacterial iron uptake.

    Directory of Open Access Journals (Sweden)

    Qingping Xu

    Full Text Available Imelysin-like proteins define a superfamily of bacterial proteins that are likely involved in iron uptake. Members of this superfamily were previously thought to be peptidases and were included in the MEROPS family M75. We determined the first crystal structures of two remotely related, imelysin-like proteins. The Psychrobacter arcticus structure was determined at 2.15 Å resolution and contains the canonical imelysin fold, while higher resolution structures from the gut bacteria Bacteroides ovatus, in two crystal forms (at 1.25 Å and 1.44 Å resolution, have a circularly permuted topology. Both structures are highly similar to each other despite low sequence similarity and circular permutation. The all-helical structure can be divided into two similar four-helix bundle domains. The overall structure and the GxHxxE motif region differ from known HxxE metallopeptidases, suggesting that imelysin-like proteins are not peptidases. A putative functional site is located at the domain interface. We have now organized the known homologous proteins into a superfamily, which can be separated into four families. These families share a similar functional site, but each has family-specific structural and sequence features. These results indicate that imelysin-like proteins have evolved from a common ancestor, and likely have a conserved function.

  19. Genome sequence, comparative analysis and haplotype structure of the domestic dog.

    Science.gov (United States)

    Lindblad-Toh, Kerstin; Wade, Claire M; Mikkelsen, Tarjei S; Karlsson, Elinor K; Jaffe, David B; Kamal, Michael; Clamp, Michele; Chang, Jean L; Kulbokas, Edward J; Zody, Michael C; Mauceli, Evan; Xie, Xiaohui; Breen, Matthew; Wayne, Robert K; Ostrander, Elaine A; Ponting, Chris P; Galibert, Francis; Smith, Douglas R; DeJong, Pieter J; Kirkness, Ewen; Alvarez, Pablo; Biagi, Tara; Brockman, William; Butler, Jonathan; Chin, Chee-Wye; Cook, April; Cuff, James; Daly, Mark J; DeCaprio, David; Gnerre, Sante; Grabherr, Manfred; Kellis, Manolis; Kleber, Michael; Bardeleben, Carolyne; Goodstadt, Leo; Heger, Andreas; Hitte, Christophe; Kim, Lisa; Koepfli, Klaus-Peter; Parker, Heidi G; Pollinger, John P; Searle, Stephen M J; Sutter, Nathan B; Thomas, Rachael; Webber, Caleb; Baldwin, Jennifer; Abebe, Adal; Abouelleil, Amr; Aftuck, Lynne; Ait-Zahra, Mostafa; Aldredge, Tyler; Allen, Nicole; An, Peter; Anderson, Scott; Antoine, Claudel; Arachchi, Harindra; Aslam, Ali; Ayotte, Laura; Bachantsang, Pasang; Barry, Andrew; Bayul, Tashi; Benamara, Mostafa; Berlin, Aaron; Bessette, Daniel; Blitshteyn, Berta; Bloom, Toby; Blye, Jason; Boguslavskiy, Leonid; Bonnet, Claude; Boukhgalter, Boris; Brown, Adam; Cahill, Patrick; Calixte, Nadia; Camarata, Jody; Cheshatsang, Yama; Chu, Jeffrey; Citroen, Mieke; Collymore, Alville; Cooke, Patrick; Dawoe, Tenzin; Daza, Riza; Decktor, Karin; DeGray, Stuart; Dhargay, Norbu; Dooley, Kimberly; Dooley, Kathleen; Dorje, Passang; Dorjee, Kunsang; Dorris, Lester; Duffey, Noah; Dupes, Alan; Egbiremolen, Osebhajajeme; Elong, Richard; Falk, Jill; Farina, Abderrahim; Faro, Susan; Ferguson, Diallo; Ferreira, Patricia; Fisher, Sheila; FitzGerald, Mike; Foley, Karen; Foley, Chelsea; Franke, Alicia; Friedrich, Dennis; Gage, Diane; Garber, Manuel; Gearin, Gary; Giannoukos, Georgia; Goode, Tina; Goyette, Audra; Graham, Joseph; Grandbois, Edward; Gyaltsen, Kunsang; Hafez, Nabil; Hagopian, Daniel; Hagos, Birhane; Hall, Jennifer; Healy, Claire; Hegarty, Ryan; Honan, Tracey; Horn, Andrea; Houde, Nathan; Hughes, Leanne; Hunnicutt, Leigh; Husby, M; Jester, Benjamin; Jones, Charlien; Kamat, Asha; Kanga, Ben; Kells, Cristyn; Khazanovich, Dmitry; Kieu, Alix Chinh; Kisner, Peter; Kumar, Mayank; Lance, Krista; Landers, Thomas; Lara, Marcia; Lee, William; Leger, Jean-Pierre; Lennon, Niall; Leuper, Lisa; LeVine, Sarah; Liu, Jinlei; Liu, Xiaohong; Lokyitsang, Yeshi; Lokyitsang, Tashi; Lui, Annie; Macdonald, Jan; Major, John; Marabella, Richard; Maru, Kebede; Matthews, Charles; McDonough, Susan; Mehta, Teena; Meldrim, James; Melnikov, Alexandre; Meneus, Louis; Mihalev, Atanas; Mihova, Tanya; Miller, Karen; Mittelman, Rachel; Mlenga, Valentine; Mulrain, Leonidas; Munson, Glen; Navidi, Adam; Naylor, Jerome; Nguyen, Tuyen; Nguyen, Nga; Nguyen, Cindy; Nguyen, Thu; Nicol, Robert; Norbu, Nyima; Norbu, Choe; Novod, Nathaniel; Nyima, Tenchoe; Olandt, Peter; O'Neill, Barry; O'Neill, Keith; Osman, Sahal; Oyono, Lucien; Patti, Christopher; Perrin, Danielle; Phunkhang, Pema; Pierre, Fritz; Priest, Margaret; Rachupka, Anthony; Raghuraman, Sujaa; Rameau, Rayale; Ray, Verneda; Raymond, Christina; Rege, Filip; Rise, Cecil; Rogers, Julie; Rogov, Peter; Sahalie, Julie; Settipalli, Sampath; Sharpe, Theodore; Shea, Terrance; Sheehan, Mechele; Sherpa, Ngawang; Shi, Jianying; Shih, Diana; Sloan, Jessie; Smith, Cherylyn; Sparrow, Todd; Stalker, John; Stange-Thomann, Nicole; Stavropoulos, Sharon; Stone, Catherine; Stone, Sabrina; Sykes, Sean; Tchuinga, Pierre; Tenzing, Pema; Tesfaye, Senait; Thoulutsang, Dawa; Thoulutsang, Yama; Topham, Kerri; Topping, Ira; Tsamla, Tsamla; Vassiliev, Helen; Venkataraman, Vijay; Vo, Andy; Wangchuk, Tsering; Wangdi, Tsering; Weiand, Michael; Wilkinson, Jane; Wilson, Adam; Yadav, Shailendra; Yang, Shuli; Yang, Xiaoping; Young, Geneva; Yu, Qing; Zainoun, Joanne; Zembek, Lisa; Zimmer, Andrew; Lander, Eric S

    2005-12-08

    Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.

  20. Optical properties of structures composed of periodic, quasi-periodic, and aperiodic sequences of particulate monolayers

    Science.gov (United States)

    Loiko, V. A.; Miskevich, A. A.

    2017-01-01

    The spectra of the coherent transmission and reflection coefficients of multilayers consisting of the periodic, Fibonacci (quasi-periodic), and Thue-Morse (aperiodic) sequences of plane-parallel monolayers of monodisperse spherical alumina and silica particles are investigated using the quasi-crystalline approximation (QCA) and the transfer matrix method (TMM). The additional opportunities for the transmission and reflection spectra manipulation in comparison with the periodic sequence of monolayers are demonstrated. Photonic band gaps in the spectra of the particulate structures are shifted to the short-wavelength range in comparison with those for systems of homogeneous layers. The shift is larger for the Thue-Morse sequence. The widths of the photonic band gaps for particulate systems are narrower than the ones for multilayers consisting of homogeneous layers of an equivalent volume of matter. The results can be used to create optical, optoelectronics, and photonics devices—for example, multispectral filters, light emitting diodes, solar cells, displays.

  1. Multi-scale coding of genomic information: From DNA sequence to genome structure and function

    Energy Technology Data Exchange (ETDEWEB)

    Arneodo, Alain, E-mail: alain.arneodo@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Vaillant, Cedric, E-mail: cedric.vaillant@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Audit, Benjamin, E-mail: benjamin.audit@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Argoul, Francoise, E-mail: francoise.argoul@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); D' Aubenton-Carafa, Yves, E-mail: daubenton@cgm.cnrs-gif.f [Centre de Genetique Moleculaire, CNRS, Allee de la Terrasse, 91198 Gif-sur-Yvette (France); Thermes, Claude, E-mail: claude.thermes@cgm.cnrs-gif.f [Centre de Genetique Moleculaire, CNRS, Allee de la Terrasse, 91198 Gif-sur-Yvette (France)

    2011-02-15

    Understanding how chromatin is spatially and dynamically organized in the nucleus of eukaryotic cells and how this affects genome functions is one of the main challenges of cell biology. Since the different orders of packaging in the hierarchical organization of DNA condition the accessibility of DNA sequence elements to trans-acting factors that control the transcription and replication processes, there is actually a wealth of structural and dynamical information to learn in the primary DNA sequence. In this review, we show that when using concepts, methodologies, numerical and experimental techniques coming from statistical mechanics and nonlinear physics combined with wavelet-based multi-scale signal processing, we are able to decipher the multi-scale sequence encoding of chromatin condensation-decondensation mechanisms that play a fundamental role in regulating many molecular processes involved in nuclear functions.

  2. A distributed computational search strategy for the identification of diagnostics targets: application to finding aptamer targets for methicillin-resistant staphylococci.

    Science.gov (United States)

    Flanagan, Keith; Cockell, Simon; Harwood, Colin; Hallinan, Jennifer; Nakjang, Sirintra; Lawry, Beth; Wipat, Anil

    2014-06-30

    The rapid and cost-effective identification of bacterial species is crucial, especially for clinical diagnosis and treatment. Peptide aptamers have been shown to be valuable for use as a component of novel, direct detection methods. These small peptides have a number of advantages over antibodies, including greater specificity and longer shelf life. These properties facilitate their use as the detector components of biosensor devices. However, the identification of suitable aptamer targets for particular groups of organisms is challenging. We present a semi-automated processing pipeline for the identification of candidate aptamer targets from whole bacterial genome sequences. The pipeline can be configured to search for protein sequence fragments that uniquely identify a set of strains of interest. The system is also capable of identifying additional organisms that may be of interest due to their possession of protein fragments in common with the initial set. Through the use of Cloud computing technology and distributed databases, our system is capable of scaling with the rapidly growing genome repositories, and consequently of keeping the resulting data sets up-to-date. The system described is also more generically applicable to the discovery of specific targets for other diagnostic approaches such as DNA probes, PCR primers and antibodies.

  3. 3DFlu: database of sequence and structural variability of the influenza hemagglutinin at population scale

    Science.gov (United States)

    Mazzocco, Giovanni; Lazniewski, Michal; Migdał, Piotr; Szczepińska, Teresa; Radomski, Jan P.; Plewczynski, Dariusz

    2016-01-01

    The influenza virus type A (IVA) is an important pathogen which is able to cause annual epidemics and even pandemics. This fact is the consequence of the antigenic shifts and drifts capabilities of IVA, caused by the high mutation rate and the reassortment capabilities of the virus. The hemagglutinin (HA) protein constitutes the main IVA antigen and has a crucial role in the infection mechanism, being responsible for the recognition of host-specific sialic acid derivatives. Despite the relative abundance of HA sequence and serological studies, comparative structure-based analysis of HA are less investigated. The 3DFlu database contains well annotated HA representatives: 1192 models and 263 crystallographic structures. The relations between these proteins are defined using different metrics and are visualized as a network in the provided web interface. Moreover structural and sequence comparison of the proteins can be explored. Metadata information (e.g. protein identifier, IVA strain, year and location of infection) can enhance the exploration of the presented data. With our database researchers gain a useful tool for the exploration of high quality HA models, viewing and comparing changes in the HA viral subtypes at several information levels (sequence, structure, ESP). The complete and integrated view of those relations might be useful to determine the efficiency of transmission, pathogenicity and for the investigation of evolutionary tendencies of the influenza virus. Database URL: http://nucleus3d.cent.uw.edu.pl/influenza PMID:27694207

  4. Identification of selectivity determinants in CYP monooxygenases by modelling and systematic analysis of sequence and structure.

    Science.gov (United States)

    Seifert, Alexander; Pleiss, Jurgen

    2012-02-01

    Cytochrome P450 monooxygenases (CYPs) form a large, ubiquitous enzyme family and are of great interest in red and white biotechnology. To investigate the effect of protein structure on selectivity, the binding of substrate molecules near to the active site was modelled by molecular dynamics simulations. From a comprehensive and systematic comparison of more than 6300 CYP sequences and 31 structures using the Cytochrome P450 Engineering Database (CYPED), residues were identified which are predicted to point close to the heme centre and thus restrict accessibility for substrates. As a result, sequence-structure-function relationships are described that can be used to predict selectivity-determining positions from CYP sequences and structures. Based on this analysis, a minimal library consisting of bacterial CYP102A1 (P450(BM3)) and 24 variants was constructed. All variants were functionally expressed in E. coli, and the library was screened with four terpene substrates. Only 3 variants showed no activity towards all 4 terpenes, while 11 variants demonstrated either a strong shift or improved regio- or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type. The minimal library also contains variants that show interesting side products which are not generated by the wild type enzyme. By two additional rounds of molecular modelling, diversification, and screening, the selectivity of one of these variants for a new product was optimised with a minimal screening effort. We propose this as a generic approach for other CYP substrates.

  5. 赭曲霉毒素A核酸适配体筛选%Screening of DNA aptamer of ochratoxin A

    Institute of Scientific and Technical Information of China (English)

    刘继红; 胡颖; 洪慧杰; 张玲; 王红旗

    2015-01-01

    Objective To screen aptamer of ochratoxin A (OTA) in wheat and other related food. Methods The solid-target selection strategy and magnetic-separation selection method were adopted to screen aptamer of ochratoxin A. Results The OTA aptamer had high affinity with nanomolar dissociation constants and good specificity non-recognizing, and it had weak affinity towards structure similarity compounds of OTA such as Ochratoxin B (OTB), N-acetyl-L-phenylalanine and warfarin. Conclusion The OTA aptamer that got from solid-target selection strategy and magnetic-separation selection method, has high affinity and good specificity. Instead of traditional antibody, the OTA aptamer may be used to develop sensor and affinity chromatography column for mycotoxins rapid detection in the field of agricultural products.%目的:筛选小麦和其他相关食品中常见的赭曲霉毒素 A 真菌毒素核酸适配体。方法采用固态靶标筛选策略和磁分离的筛选方法筛选赭曲霉毒素A的核酸适配体。结果获得的赭曲霉毒素A核酸适配体亲和力较高,解离常数为纳摩尔级;特异性好,与赭曲霉毒素A结构相似性化合物如赭曲霉毒素B、N-乙酰苯丙氨酸或华法令不结合或结合力非常弱。结论本文采用固态靶标筛选策略和磁分离的SELEX筛选方法获得了赭曲霉毒素 A 的高特异性、高亲和力核酸适配体,有望在农产品质量安全领域替代传统抗体开发真菌毒素快速检测传感器或制备固相亲和柱。

  6. Multiple amino acid sequence alignment nitrogenase component 1: insights into phylogenetics and structure-function relationships.

    Directory of Open Access Journals (Sweden)

    James B Howard

    Full Text Available Amino acid residues critical for a protein's structure-function are retained by natural selection and these residues are identified by the level of variance in co-aligned homologous protein sequences. The relevant residues in the nitrogen fixation Component 1 α- and β-subunits were identified by the alignment of 95 protein sequences. Proteins were included from species encompassing multiple microbial phyla and diverse ecological niches as well as the nitrogen fixation genotypes, anf, nif, and vnf, which encode proteins associated with cofactors differing at one metal site. After adjusting for differences in sequence length, insertions, and deletions, the remaining >85% of the sequence co-aligned the subunits from the three genotypes. Six Groups, designated Anf, Vnf , and Nif I-IV, were assigned based upon genetic origin, sequence adjustments, and conserved residues. Both subunits subdivided into the same groups. Invariant and single variant residues were identified and were defined as "core" for nitrogenase function. Three species in Group Nif-III, Candidatus Desulforudis audaxviator, Desulfotomaculum kuznetsovii, and Thermodesulfatator indicus, were found to have a seleno-cysteine that replaces one cysteinyl ligand of the 8Fe:7S, P-cluster. Subsets of invariant residues, limited to individual groups, were identified; these unique residues help identify the gene of origin (anf, nif, or vnf yet should not be considered diagnostic of the metal content of associated cofactors. Fourteen of the 19 residues that compose the cofactor pocket are invariant or single variant; the other five residues are highly variable but do not correlate with the putative metal content of the cofactor. The variable residues are clustered on one side of the cofactor, away from other functional centers in the three dimensional structure. Many of the invariant and single variant residues were not previously recognized as potentially critical and their identification

  7. Timing of developmental sequences in different brain structures: physiological and pathological implications.

    Science.gov (United States)

    Dehorter, N; Vinay, L; Hammond, C; Ben-Ari, Y

    2012-06-01

    The developing brain is not a small adult brain. Voltage- and transmitter-gated currents, like network-driven patterns, follow a developmental sequence. Studies initially performed in cortical structures and subsequently in subcortical structures have unravelled a developmental sequence of events in which intrinsic voltage-gated calcium currents are followed by nonsynaptic calcium plateaux and synapse-driven giant depolarising potentials, orchestrated by depolarizing actions of GABA and long-lasting NMDA receptor-mediated currents. The function of these early patterns is to enable heterogeneous neurons to fire and wire together rather than to code specific modalities. However, at some stage, behaviourally relevant activities must replace these immature patterns, implying the presence of programmed stop signals. Here, we show that the developing striatum follows a developmental sequence in which immature patterns are silenced precisely when the pup starts locomotion. This is mediated by a loss of the long-lasting NMDA-NR2C/D receptor-mediated current and the expression of a voltage-gated K(+) current. At the same time, the descending inputs to the spinal cord become fully functional, accompanying a GABA/glycine polarity shift and ending the expression of developmental patterns. Therefore, although the timetable of development differs in different brain structures, the g sequence is quite similar, relying first on nonsynaptic events and then on synaptic oscillations that entrain large neuronal populations. In keeping with the 'neuroarcheology' theory, genetic mutations or environmental insults that perturb these developmental sequences constitute early signatures of developmental disorders. Birth dating developmental disorders thus provides important indicators of the event that triggers the pathological cascade leading ultimately to disease.

  8. Multiple amino acid sequence alignment nitrogenase component 1: insights into phylogenetics and structure-function relationships.

    Science.gov (United States)

    Howard, James B; Kechris, Katerina J; Rees, Douglas C; Glazer, Alexander N

    2013-01-01

    Amino acid residues critical for a protein's structure-function are retained by natural selection and these residues are identified by the level of variance in co-aligned homologous protein sequences. The relevant residues in the nitrogen fixation Component 1 α- and β-subunits were identified by the alignment of 95 protein sequences. Proteins were included from species encompassing multiple microbial phyla and diverse ecological niches as well as the nitrogen fixation genotypes, anf, nif, and vnf, which encode proteins associated with cofactors differing at one metal site. After adjusting for differences in sequence length, insertions, and deletions, the remaining >85% of the sequence co-aligned the subunits from the three genotypes. Six Groups, designated Anf, Vnf , and Nif I-IV, were assigned based upon genetic origin, sequence adjustments, and conserved residues. Both subunits subdivided into the same groups. Invariant and single variant residues were identified and were defined as "core" for nitrogenase function. Three species in Group Nif-III, Candidatus Desulforudis audaxviator, Desulfotomaculum kuznetsovii, and Thermodesulfatator indicus, were found to have a seleno-cysteine that replaces one cysteinyl ligand of the 8Fe:7S, P-cluster. Subsets of invariant residues, limited to individual groups, were identified; these unique residues help identify the gene of origin (anf, nif, or vnf) yet should not be considered diagnostic of the metal content of associated cofactors. Fourteen of the 19 residues that compose the cofactor pocket are invariant or single variant; the other five residues are highly variable but do not correlate with the putative metal content of the cofactor. The variable residues are clustered on one side of the cofactor, away from other functional centers in the three dimensional structure. Many of the invariant and single variant residues were not previously recognized as potentially critical and their identification provides the bases for

  9. ERP analysis of cognitive sequencing: a left anterior negativity related to structural transformation processing.

    Science.gov (United States)

    Hoen, M; Dominey, P F

    2000-09-28

    A major objective of cognitive neuroscience is to identify those neurocomputational processes that may be shared by multiple cognitive functions vs those that are highly specific. This problem of identifying general vs specialized functions is of particular interest in the domain of language processing. Within this domain, event related brain potential (ERP) studies have demonstrated a left anterior negativity (LAN) in a range 300-700 ms, associated with syntactic processing, often linked to grammatical function words. These words have little or no semantic content, but rather play a role in encoding syntactic structure required for parsing. In the current study we test the hypothesis that the LAN reflects the operation of a more general sequence processing capability in which special symbols encode structural information that, when combined with past elements in the sequence, allows the prediction of successor elements. We recorded ERPs during a non-linguistic sequencing task that required subjects (n = 10) to process special symbols possessing the functional property defined above. When compared to ERPs in a control condition, function symbol processing elicits a left anterior negative shift between temporal and spatial characteristics quite similar to the LAN described during function word processing in language, supporting our hypothesis. These results are discussed in the context of related studies of syntactic and cognitive sequence processing.

  10. An anatomically structured sensory-motor sequence learning system displays some general linguistic capacities.

    Science.gov (United States)

    Dominey, P F

    1997-08-01

    The capacity in primates to master temporal-sequential constraints from the external world might provide a basis for accommodating similar constraints in language. While the neural specialization required for language clearly distinguishes man from the other primates, it is less clear to what extent this specialization constitutes a drastic neurophysiological divergence versus a variation on an existing sequencing capability. In an effort to address this issue, an anatomically structured neural network model, previously developed to reproduce complex sensory-motor sequences and the corresponding single-unit recordings from primate prefrontal cortex, is studied in a simple linguistic context. The model is presented sentences from a small language and demonstrates a simple capacity to "understand" and generalize at different levels. Interactions between variations on (a) the model architecture and (b) the target language structure agree with data from crosslinguistic aphasia studies. These results support the hypothesis that a brain architecture for nonlinguistic cognitive functions (in this case sensory-motor sequencing) can provide a basis for a general sequence processing component of linguistic function.

  11. Comparison of sequence-based and structure-based phylogenetic trees of homologous proteins: Inferences on protein evolution

    Indian Academy of Sciences (India)

    S Balaji; N Srinivasan

    2007-01-01

    Several studies based on the known three-dimensional (3-D) structures of proteins show that two homologous proteins with insignificant sequence similarity could adopt a common fold and may perform same or similar biochemical functions. Hence, it is appropriate to use similarities in 3-D structure of proteins rather than the amino acid sequence similarities in modelling evolution of distantly related proteins. Here we present an assessment of using 3-D structures in modelling evolution of homologous proteins. Using a dataset of 108 protein domain families of known structures with at least 10 members per family we present a comparison of extent of structural and sequence dissimilarities among pairs of proteins which are inputs into the construction of phylogenetic trees. We find that correlation between the structure-based dissimilarity measures and the sequence-based dissimilarity measures is usually good if the sequence similarity among the homologues is about 30% or more. For protein families with low sequence similarity among the members, the correlation coefficient between the sequence-based and the structure-based dissimilarities are poor. In these cases the structure-based dendrogram clusters proteins with most similar biochemical functional properties better than the sequence-similarity based dendrogram. In multi-domain protein families and disulphide-rich protein families the correlation coefficient for the match of sequence-based and structure-based dissimilarity (SDM) measures can be poor though the sequence identity could be higher than 30%. Hence it is suggested that protein evolution is best modelled using 3-D structures if the sequence similarities (SSM) of the homologues are very low.

  12. Structural insights into DNA sequence recognition by Type ISP restriction-modification enzymes.

    Science.gov (United States)

    Kulkarni, Manasi; Nirwan, Neha; van Aelst, Kara; Szczelkun, Mark D; Saikrishnan, Kayarat

    2016-05-19

    Engineering restriction enzymes with new sequence specificity has been an unaccomplished challenge, presumably because of the complexity of target recognition. Here we report detailed analyses of target recognition by Type ISP restriction-modification enzymes. We determined the structure of the Type ISP enzyme LlaGI bound to its target and compared it with the previously reported structure of a close homologue that binds to a distinct target, LlaBIII. The comparison revealed that, although the two enzymes use almost a similar set of structural elements for target recognition, the residues that read the bases vary. Change in specificity resulted not only from appropriate substitution of amino acids that contacted the bases but also from new contacts made by positionally distinct residues directly or through a water bridge. Sequence analyses of 552 Type ISP enzymes showed that the structural elements involved in target recognition of LlaGI and LlaBIII were structurally well-conserved but sequentially less-conserved. In addition, the residue positions within these structural elements were under strong evolutionary constraint, highlighting the functional importance of these regions. The comparative study helped decipher a partial consensus code for target recognition by Type ISP enzymes.

  13. Crystal structure of pseudouridine synthase RluA: indirect sequence readout through protein-induced RNA structure.

    Science.gov (United States)

    Hoang, Charmaine; Chen, Junjun; Vizthum, Caroline A; Kandel, Jason M; Hamilton, Christopher S; Mueller, Eugene G; Ferré-D'Amaré, Adrian R

    2006-11-17

    RluA is a dual-specificity enzyme responsible for pseudouridylating 23S rRNA and several tRNAs. The 2.05 A resolution structure of RluA bound to a substrate RNA comprising the anticodon stem loop of tRNA(Phe) reveals that enzyme binding induces a dramatic reorganization of the RNA. Instead of adopting its canonical U turn conformation, the anticodon loop folds into a new structure with a reverse-Hoogsteen base pair and three flipped-out nucleotides. Sequence conservation, the cocrystal structure, and the results of structure-guided mutagenesis suggest that RluA recognizes its substrates indirectly by probing RNA loops for their ability to adopt the reorganized fold. The planar, cationic side chain of an arginine intercalates between the reverse-Hoogsteen base pair and the bottom pair of the anticodon stem, flipping the nucleotide to be modified into the active site of RluA. Sequence and structural comparisons suggest that pseudouridine synthases of the RluA, RsuA, and TruA families employ an equivalent arginine for base flipping.

  14. Prediction of protein structural features from sequence data based on Shannon entropy and Kolmogorov complexity.

    Science.gov (United States)

    Bywater, Robert Paul

    2015-01-01

    While the genome for a given organism stores the information necessary for the organism to function and flourish it is the proteins that are encoded by the genome that perhaps more than anything else characterize the phenotype for that organism. It is therefore not surprising that one of the many approaches to understanding and predicting protein folding and properties has come from genomics and more specifically from multiple sequence alignments. In this work I explore ways in which data derived from sequence alignment data can be used to investigate in a predictive way three different aspects of protein structure: secondary structures, inter-residue contacts and the dynamics of switching between different states of the protein. In particular the use of Kolmogorov complexity has identified a novel pathway towards achieving these goals.

  15. Screening and characterization of aptamers of chronic myelognous leukemia K562 cells%慢性髓细胞性白血病K562细胞适体的筛选与鉴定

    Institute of Scientific and Technical Information of China (English)

    刘玲玲; 韩跃武; 祝凯华; 李真真; 韩亚萍; 路艳; 王春霞

    2009-01-01

    目的:筛选并鉴定出慢性髓细胞性白血病(CML)K562细胞的寡核苷酸适体.方法:体外合成长度为88个碱基的随机单链DNA(ssDNA)文库,采用生物素-链霉亲和素磁珠法制备次级文库,以正常人血液中提取的中性粒细胞为反筛细胞,利用指数富集的配基系统进化(SELEX)技术筛选出与CML K562细胞特异结合的适体.将筛选得到的适体回收纯化后连接pGEM-T质粒载体,经蓝白筛选后,随机挑选24个克隆子进行序列测定.采用荧光标记引物法检测ssDNA文库与K562细胞的亲和力,并用Clustal 2.05和DNA sis V 2.5软件对适体序列进行一级结构同源性分析和二级结构预测.结果:经过13轮循环筛选,CML K562细胞适体的A值从0.12上升到1.25,至第13轮A值无明显增高.一级结构分析无同源序列,但可分为6个家族,其中5个家族各自具有保守序列,家族6无保守序列.二级结构分析表明,适体形成的茎环、凸环结构可能是与K562细胞特异性结合的结构基础.结论:利用SELEX技术成功筛选出高亲和性的CML K562细胞适体.%AIM: To screen and characterize oligonucleotide aptamers of chronic myelognous leukemia K562 cells. METHODS: Oligonucleotide aptamers specifically binding to chronic myelog-nous leukemia K562 cells were screened from 88 nt random ssDNA library in vitro syntbesis by SELEX method, sub-library was prepared by biotin-streptavidin magnetic beads and neutro-phils from blood of normal humans were used as anti-sieve cells. The screened aptamters were purified and connected to pGEM-T plasmid vector and 24 clones of random aelection were sequenced after screening by the blue and white. The affinities of the screened aptamers binding to chronic myelognous leukemia K562 cells were detected by fluorescent primers. Homology analyses of the primary structure and secondary structure prediction to the screened aptamters were conducted with Clustal 2.05 and DNA sis V 2.5 software. RESULTS: After

  16. Evol and ProDy for bridging protein sequence evolution and structural dynamics

    OpenAIRE

    Bakan, Ahmet; Dutta, Anindita; Mao, Wenzhi; Liu, Ying; Chennubhotla, Chakra; Lezon, Timothy R.; Bahar, Ivet

    2014-01-01

    Correlations between sequence evolution and structural dynamics are of utmost importance in understanding the molecular mechanisms of function and their evolution. We have integrated Evol, a new package for fast and efficient comparative analysis of evolutionary patterns and conformational dynamics, into ProDy, a computational toolbox designed for inferring protein dynamics from experimental and theoretical data. Using information-theoretic approaches, Evol coanalyzes conservation and coevolu...

  17. Revised Mimivirus major capsid protein sequence reveals intron-containing gene structure and extra domain

    Directory of Open Access Journals (Sweden)

    Suzan-Monti Marie

    2009-05-01

    Full Text Available Abstract Background Acanthamoebae polyphaga Mimivirus (APM is the largest known dsDNA virus. The viral particle has a nearly icosahedral structure with an internal capsid shell surrounded with a dense layer of fibrils. A Capsid protein sequence, D13L, was deduced from the APM L425 coding gene and was shown to be the most abundant protein found within the viral particle. However this protein remained poorly characterised until now. A revised protein sequence deposited in a database suggested an additional N-terminal stretch of 142 amino acids missing from the original deduced sequence. This result led us to investigate the L425 gene structure and the biochemical properties of the complete APM major Capsid protein. Results This study describes the full length 3430 bp Capsid coding gene and characterises the 593 amino acids long corresponding Capsid protein 1. The recombinant full length protein allowed the production of a specific monoclonal antibody able to detect the Capsid protein 1 within the viral particle. This protein appeared to be post-translationnally modified by glycosylation and phosphorylation. We proposed a secondary structure prediction of APM Capsid protein 1 compared to the Capsid protein structure of Paramecium Bursaria Chlorella Virus 1, another member of the Nucleo-Cytoplasmic Large DNA virus family. Conclusion The characterisation of the full length L425 Capsid coding gene of Acanthamoebae polyphaga Mimivirus provides new insights into the structure of the main Capsid protein. The production of a full length recombinant protein will be useful for further structural studies.

  18. Fifty Years Later: The Sequence, Structure and Function of Lacewing Cross-beta Silk

    Energy Technology Data Exchange (ETDEWEB)

    Weisman, Sarah; Okada, Shoko; Mudie, Stephen T.; Huson, Mickey G.; Trueman, Holly E.; Sriskantha, Alagacone; Haritos, Victoria S.; Sutherland, Tara D.; (CSIRO/MSE); (CSIRO)

    2009-12-01

    Classic studies of protein structure in the 1950s and 1960s demonstrated that green lacewing egg stalk silk possesses a rare native cross-beta sheet conformation. We have identified and sequenced the silk genes expressed by adult females of a green lacewing species. The two encoded silk proteins are 109 and 67 kDa in size and rich in serine, glycine and alanine. Over 70% of each protein sequence consists of highly repetitive regions with 16-residue periodicity. The repetitive sequences can be fitted to an elegant cross-beta sheet structural model with protein chains folded into regular 8-residue long beta strands. This model is supported by wide-angle X-ray scattering data and tensile testing from both our work and the original papers. We suggest that the silk proteins assemble into stacked beta sheet crystallites bound together by a network of cystine cross-links. This hierarchical structure gives the lacewing silk high lateral stiffness nearly threefold that of silkworm silk, enabling the egg stalks to effectively suspend eggs and protect them from predators.

  19. Interpolation on a fixed interval discrete-valued sequence with random structure

    Directory of Open Access Journals (Sweden)

    D. H. Ilyasova

    2011-06-01

    Full Text Available Discrete-valued sequences with random structure are widely used to describe electronic systems that operate under a priori uncertainty. An optimal interpolation algorithm on a fixed interval discrete-valued sequence with random structure have been obtained considering the Markov property of an extended process, which includes the value of a discrete-type sequence and its structure. This algorithm is recursive, and describes the evolution of the joint interpolation probability of the extended process in reverse time. Analysis of the optimal interpolation algorithm on a fixed interval was implemented by the example of decoding of a convolutional code by means of statistical computer modeling. For this example interpolation algorithm reduces the bit error probability to 3-4 times compared with the algorithm of filtering due to the fact that it takes into account all the received observations. The increase of statistical dependence between input symbols leads to a decrease in bit error rate in filtration and interpolation algorithms.

  20. SVM-PB-Pred: SVM based protein block prediction method using sequence profiles and secondary structures.

    Science.gov (United States)

    Suresh, V; Parthasarathy, S

    2014-01-01

    We developed a support vector machine based web server called SVM-PB-Pred, to predict the Protein Block for any given amino acid sequence. The input features of SVM-PB-Pred include i) sequence profiles (PSSM) and ii) actual secondary structures (SS) from DSSP method or predicted secondary structures from NPS@ and GOR4 methods. There were three combined input features PSSM+SS(DSSP), PSSM+SS(NPS@) and PSSM+SS(GOR4) used to test and train the SVM models. Similarly, four datasets RS90, DB433, LI1264 and SP1577 were used to develop the SVM models. These four SVM models developed were tested using three different benchmarking tests namely; (i) self consistency, (ii) seven fold cross validation test and (iii) independent case test. The maximum possible prediction accuracy of ~70% was observed in self consistency test for the SVM models of both LI1264 and SP1577 datasets, where PSSM+SS(DSSP) input features was used to test. The prediction accuracies were reduced to ~53% for PSSM+SS(NPS@) and ~43% for PSSM+SS(GOR4) in independent case test, for the SVM models of above two same datasets. Using our method, it is possible to predict the protein block letters for any query protein sequence with ~53% accuracy, when the SP1577 dataset and predicted secondary structure from NPS@ server were used. The SVM-PB-Pred server can be freely accessed through http://bioinfo.bdu.ac.in/~svmpbpred.

  1. Rupture of DNA aptamer: New insights from simulations

    Energy Technology Data Exchange (ETDEWEB)

    Mishra, Rakesh Kumar; Nath, Shesh; Kumar, Sanjay [Department of Physics, Banaras Hindu University, Varanasi 221 005 (India)

    2015-10-28

    Base-pockets (non-complementary base-pairs) in a double-stranded DNA play a crucial role in biological processes. Because of thermal fluctuations, it can lower the stability of DNA, whereas, in case of DNA aptamer, small molecules, e.g., adenosinemonophosphate and adenosinetriphosphate, form additional hydrogen bonds with base-pockets termed as “binding-pockets,” which enhance the stability. Using the Langevin dynamics simulations of coarse grained model of DNA followed by atomistic simulations, we investigated the influence of base-pocket and binding-pocket on the stability of DNA aptamer. Striking differences have been reported here for the separation induced by temperature and force, which require further investigation by single molecule experiments.

  2. Population genetic structure and historical demography of Oratosquilla oratoria revealed by mitochondrial DNA sequences.

    Science.gov (United States)

    Zhang, D; Ding, Ge; Ge, B; Zhang, H; Tang, B

    2012-12-01

    Genetic diversity, population genetic structure and molecular phylogeographic pattern of mantis shrimp Oratosquilla oratoria in Bohai Sea and South China Sea were analyzed by mitochondrial DNA sequences. Nucleotide and haplotype diversities were 0.00409-0.00669 and 0.894-0.953 respectively. Neighbor-Joining phylogenetic tree clustered two distinct lineages. Both phylogenetic tree and median-joining network showed the consistent genetic structure corresponding to geographical distribution. Mismatch distributions, negative neutral test and "star-like" network supported a sudden population expansion event. And the time was estimated about 44000 and 50000 years ago.

  3. Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins.

    Science.gov (United States)

    Hayashi, C Y; Shipley, N H; Lewis, R V

    1999-01-01

    Several types of silks and silk protein coding genes have been characterized from orb-web weaving spiders. When the protein sequences of major ampullate, minor ampullate, and flagelliform silks from Nephila clavipes are compared, they can be summarized as sets of shared amino acid motifs. Four of these motifs and their likely secondary structures are described. Each structural element, termed a module, is then associated with its impact on the mechanical properties of a silk fiber. In particular, correlations are drawn between an alanine-rich 'crystalline module' and tensile strength and between a proline-containing 'elasticity module' and extensibility.

  4. Using atomic force microscopy and surface plasmon resonance to detect specific interactions between ricin and anti-ricin aptamers

    Science.gov (United States)

    Nucleic acid aptamers have been widely used as binding reagents for the label free detections of biomolecules. Compare to antibodies, aptamers have demonstrated advantages such as easy synthesis, low cost, and better stability. Therefore, aptamers can be integrated into various detection platforms ...

  5. Smart ligand: aptamer-mediated targeted delivery of chemotherapeutic drugs and siRNA for cancer therapy.

    Science.gov (United States)

    Li, Xin; Zhao, Qinghe; Qiu, Liyan

    2013-10-28

    Aptamers are a class of oligonucleotides that can specifically bind to different targets with high affinity. Since their discovery in 1980s, aptamers have attracted considerable interests in medical applications. So far, initial research using aptamers as delivery systems has produced exciting results. In this review, we summarize recent progress in aptamer-mediated chemotherapeutic drug and siRNA delivery systems in tumor treatment. With regard to chemotherapeutic drugs, the 2 main methods for targeted delivery using aptamers are as follows: aptamer-drug systems (in which aptamers directly deliver the drug both as a carrier and as a ligand) and aptamer-nanoparticles systems (in which nanoparticles function together with aptamers for targeted delivery of drugs). For delivery of siRNA, aptamers can be utilized by the following ways to facilitate targeting: (1) linked by a connector; (2) form a chimera; and (3) combined with nanoparticles. In co-delivery system, the advantages associated with the use of aptamers are beginning to become apparent also. Here, the challenges and new perspectives in the field of aptamer-mediated delivery have been discussed.

  6. The four ingredients of single-sequence RNA secondary structure prediction. A unifying perspective.

    Science.gov (United States)

    Rivas, Elena

    2013-07-01

    Any method for RNA secondary structure prediction is determined by four ingredients. The architecture is the choice of features implemented by the model (such as stacked basepairs, loop length distributions, etc.). The architecture determines the number of parameters in the model. The scoring scheme is the nature of those parameters (whether thermodynamic, probabilistic, or weights). The parameterization stands for the specific values assigned to the parameters. These three ingredients are referred to as "the model." The fourth ingredient is the folding algorithms used to predict plausible secondary structures given the model and the sequence of a structural RNA. Here, I make several unifying observations drawn from looking at more than 40 years of methods for RNA secondary structure prediction in the light of this classification. As a final observation, there seems to be a performance ceiling that affects all methods with complex architectures, a ceiling that impacts all scoring schemes with remarkable similarity. This suggests that modeling RNA secondary structure by using intrinsic sequence-based plausible "foldability" will require the incorporation of other forms of information in order to constrain the folding space and to improve prediction accuracy. This could give an advantage to probabilistic scoring systems since a probabilistic framework is a natural platform to incorporate different sources of information into one single inference problem.

  7. Pattern matching through Chaos Game Representation: bridging numerical and discrete data structures for biological sequence analysis

    Directory of Open Access Journals (Sweden)

    Vinga Susana

    2012-05-01

    Full Text Available Abstract Background Chaos Game Representation (CGR is an iterated function that bijectively maps discrete sequences into a continuous domain. As a result, discrete sequences can be object of statistical and topological analyses otherwise reserved to numerical systems. Characteristically, CGR coordinates of substrings sharing an L-long suffix will be located within 2-L distance of each other. In the two decades since its original proposal, CGR has been generalized beyond its original focus on genomic sequences and has been successfully applied to a wide range of problems in bioinformatics. This report explores the possibility that it can be further extended to approach algorithms that rely on discrete, graph-based representations. Results The exploratory analysis described here consisted of selecting foundational string problems and refactoring them using CGR-based algorithms. We found that CGR can take the role of suffix trees and emulate sophisticated string algorithms, efficiently solving exact and approximate string matching problems such as finding all palindromes and tandem repeats, and matching with mismatches. The common feature of these problems is that they use longest common extension (LCE queries as subtasks of their procedures, which we show to have a constant time solution with CGR. Additionally, we show that CGR can be used as a rolling hash function within the Rabin-Karp algorithm. Conclusions The analysis of biological sequences relies on algorithmic foundations facing mounting challenges, both logistic (performance and analytical (lack of unifying mathematical framework. CGR is found to provide the latter and to promise the former: graph-based data structures for sequence analysis operations are entailed by numerical-based data structures produced by CGR maps, providing a unifying analytical framework for a diversity of pattern matching problems.

  8. Aptamer-functionalized gold nanoparticles as photoresponsive nanoplatform for co-drug delivery.

    Science.gov (United States)

    Shiao, Yi-Syun; Chiu, Huai-Hsuan; Wu, Pin-Hsuan; Huang, Yu-Fen

    2014-12-24

    Various platforms have been developed as innovative nanocarriers to deliver therapeutic agents to the diseased sites. Multifunctional surface modification allows an enhanced recognition and uptake of drug carriers by targeted cells. However, the development of drug resistance in some tumor cells plays a major role in the failure of chemotherapy. Drugs given in combination, called multidrug delivery approach, was designed to improve the therapeutic efficacy and has become an increasingly used strategy that is of great importance in clinical cancer treatments. In this study, aptamer-functionalized gold nanoparticles (Au NPs) have been used as a nanoplatform to codeliver two different anticancer drugs for improving the drug effectiveness. The surface of Au NPs (13 nm in diameter) was assembled with AS1411 aptamers, which tethered with 21-base pairs of (CGATCGA)3 sequence approached to the Au NPs. Both the photosensitizer 5,10,15,20-tetrakis(1-methylpyridinium-4-yl) porphyrin (TMPyP4) and the chemotherapeutic drug doxorubicin (Dox) were then physically attached to the AS1411-conjugated Au NPs (T/D:ds-NPs) and delivered to the target tumor cells such as HeLa and Dox-resistant MCF-7R cell lines. When exposed to a 632 nm light, reactive oxygen species induced by TMPyP4 molecules were generated inside the living cells, followed by cell damage. In addition, triggered release of the complementary drugs also occurred simultaneously during the photodynamic reaction. In the presence of Dox molecules, the toxicity toward the target cells was superior to individual drug treatment. Overall, a co-drug delivery platform was successfully established to improve the therapeutic efficacy in tumor cells. The improvement of the photodynamic-stimulated triggered release was enhanced, thus highly promising precise drug release in targeted drug delivery.

  9. Aptamer-Based Sensing Technology Towards Food Safety Analysis%面向食品安全分析的核酸适配体传感技术

    Institute of Scientific and Technical Information of China (English)

    梁淼; 刘锐; 苏荣欣; 齐崴; 王利兵; 何志敏

    2012-01-01

    Intense works have been undertaken towards development of analytical methods for the detection of risk factors in the food samples. Nucleic-acid aptamers, obtained through in vitro selection and amplification, are artificial single-stranded DNA or RNA sequences that could bind to a wide range of targets with extremely high specificity and stability. These advantages have motivated aptamers to find broad applications towards food safety analysis. In recent years, the combination of aptamers with novel nanomaterials has significantly improved the performance of aptamer-based analytical techniques. In this review, we summarize the recent development of aptamer-based sensors and detection methods, including optical, electrochemical and surface plasmon resonance methods, and highlight their potential application in food safety analysis. Also, the future perspectives of aptamer- based analytical techniques are concluded.%食品中危害因子的检测一直是国内外食品安全领域致力解决的重要问题。核酸适配体是一类通过体外筛选技术得到的单链DNA或RNA,具有特异性强、稳定性好和靶分子广等特点,因而被广泛用于食品安全检测领域。近年来,随着纳米传感技术的快速发展,互补结合适配体和纳米材料的特殊性质,可实现对靶标物质的超灵敏、高选择性及快速检测。本文总结了近年来筛选的食品危害因子适配体,综述了面向食品安全检测的基于适配体传感和纳米材料修饰的分析检测技术的进展,主要包括比色法、荧光法、电化学法以及表面等离子体共振技术,并探讨了适配体传感检测所存在的问题和未来的发展趋势。

  10. HIV-P24核酸适配体的筛选%Screening of aptamers to HIV-P24

    Institute of Scientific and Technical Information of China (English)

    张宁; 毛爱红; 马瑾; 廖世奇; 张丽琼; 王晓清; 马君

    2012-01-01

    Objective To screen aptamers to HIV-P24 through SELEX technique for the diagnosis and therapy of AIDS. Methods With recombinant P24 for the screening target, oligonucleotides binding to HIV-P24 were screened from a random oligonucleotide library through SELEX technique. The binding capacity between oligonucleotides obtained from the 12th round of screening and HIV-P24 was identified via electrophoretic mobility shift assay (EMSA). The aptamers strongly binding to HIV-P24 were screened and the recognition specificity of aptamers for HIV-P24 was detected by Dot-blot method. Results Five aptamers with high affinity to HIV-P24 were obtained with different sequences. The binding specificity showed that No. 18 and No. 26 apatmers only bound to HIV-P24, not to human serum albumin, bovine serum albumin and skimmed milk powder. Conclusions Two aptamers specially binding to HIV-P24 were obtained, and thus provides an experimental basis for the diagnosis and treatment of AIDS by utilizing aptamer of HIV-P24.%目的 利用SELEX技术筛选HIV-P24的核酸适配体,为艾滋病的诊断和治疗奠定基础.方法 以重组P24为筛选靶,用SELEX技术从随机寡核苷酸库中筛选与HIV-P24结合的寡核苷酸,利用凝胶阻滞实验鉴定第12轮筛选到的寡核苷酸与HIV-P24的结合,再用Dot-blot法筛选出与HIV-P24结合的核酸适配体,并检测核酸适配体识别HIV-P24的特异性.结果 Dot-blot筛选到5条与HIV-P24有较强结合能力的核酸适配体,且均为不同的序列.特异性检测显示,18和26号配体只与HIV-P24特异性结合,而与人血清白蛋白、牛血清白蛋白和脱脂奶粉均无明显结合.结论 成功筛选到2条特异结合HIV-P24的核酸适配体,为其应用于艾滋病诊断和治疗提供了实验基础.

  11. Ribosomal DNA sequence heterogeneity reflects intraspecies phylogenies and predicts genome structure in two contrasting yeast species.

    Science.gov (United States)

    West, Claire; James, Stephen A; Davey, Robert P; Dicks, Jo; Roberts, Ian N

    2014-07-01

    The ribosomal RNA encapsulates a wealth of evolutionary information, including genetic variation that can be used to discriminate between organisms at a wide range of taxonomic levels. For example, the prokaryotic 16S rDNA sequence is very widely used both in phylogenetic studies and as a marker in metagenomic surveys and the internal transcribed spacer region, frequently used in plant phylogenetics, is now recognized as a fungal DNA barcode. However, this widespread use does not escape criticism, principally due to issues such as difficulties in classification of paralogous versus orthologous rDNA units and intragenomic variation, both of which may be significant barriers to accurate phylogenetic inference. We recently analyzed data sets from the Saccharomyces Genome Resequencing Project, characterizing rDNA sequence variation within multiple strains of the baker's yeast Saccharomyces cerevisiae and its nearest wild relative Saccharomyces paradoxus in unprecedented detail. Notably, both species possess single locus rDNA systems. Here, we use these new variation datasets to assess whether a more detailed characterization of the rDNA locus can alleviate the second of these phylogenetic issues, sequence heterogeneity, while controlling for the first. We demonstrate that a strong phylogenetic signal exists within both datasets and illustrate how they can be used, with existing methodology, to estimate intraspecies phylogenies of yeast strains consistent with those derived from whole-genome approaches. We also describe the use of partial Single Nucleotide Polymorphisms, a type of sequence variation found only in repetitive genomic regions, in identifying key evolutionary features such as genome hybridization events and show their consistency with whole-genome Structure analyses. We conclude that our approach can transform rDNA sequence heterogeneity from a problem to a useful source of evolutionary information, enabling the estimation of highly accurate phylogenies of

  12. Sequence-structure relationship study in all-α transmembrane proteins using an unsupervised learning approach.

    Science.gov (United States)

    Esque, Jérémy; Urbain, Aurélie; Etchebest, Catherine; de Brevern, Alexandre G

    2015-11-01

    Transmembrane proteins (TMPs) are major drug targets, but the knowledge of their precise topology structure remains highly limited compared with globular proteins. In spite of the difficulties in obtaining their structures, an important effort has been made these last years to increase their number from an experimental and computational point of view. In view of this emerging challenge, the development of computational methods to extract knowledge from these data is crucial for the better understanding of their functions and in improving the quality of structural models. Here, we revisit an efficient unsupervised learning procedure, called Hybrid Protein Model (HPM), which is applied to the analysis of transmembrane proteins belonging to the all-α structural class. HPM method is an original classification procedure that efficiently combines sequence and structure learning. The procedure was initially applied to the analysis of globular proteins. In the present case, HPM classifies a set of overlapping protein fragments, extracted from a non-redundant databank of TMP 3D structure. After fine-tuning of the learning parameters, the optimal classification results in 65 clusters. They represent at best similar relationships between sequence and local structure properties of TMPs. Interestingly, HPM distinguishes among the resulting clusters two helical regions with distinct hydrophobic patterns. This underlines the complexity of the topology of these proteins. The HPM classification enlightens unusual relationship between amino acids in TMP fragments, which can be useful to elaborate new amino acids substitution matrices. Finally, two challenging applications are described: the first one aims at annotating protein functions (channel or not), the second one intends to assess the quality of the structures (X-ray or models) via a new scoring function deduced from the HPM classification.

  13. High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

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    Soichi Inagaki

    Full Text Available Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

  14. Sequence-structure-function relations of the mosquito leucine-rich repeat immune proteins

    Directory of Open Access Journals (Sweden)

    Povelones Michael

    2010-09-01

    Full Text Available Abstract Background The discovery and characterisation of factors governing innate immune responses in insects has driven the elucidation of many immune system components in mammals and other organisms. Focusing on the immune system responses of the malaria mosquito, Anopheles gambiae, has uncovered an array of components and mechanisms involved in defence against pathogen infections. Two of these immune factors are LRIM1 and APL1C, which are leucine-rich repeat (LRR containing proteins that activate complement-like defence responses against malaria parasites. In addition to their LRR domains, these leucine-rich repeat immune (LRIM proteins share several structural features including signal peptides, patterns of cysteine residues, and coiled-coil domains. Results The identification and characterisation of genes related to LRIM1 and APL1C revealed putatively novel innate immune factors and furthered the understanding of their likely molecular functions. Genomic scans using the shared features of LRIM1 and APL1C identified more than 20 LRIM-like genes exhibiting all or most of their sequence features in each of three disease-vector mosquitoes with sequenced genomes: An. gambiae, Aedes aegypti, and Culex quinquefasciatus. Comparative sequence analyses revealed that this family of mosquito LRIM-like genes is characterised by a variable number of 6 to 14 LRRs of different lengths. The "Long" LRIM subfamily, with 10 or more LRRs, and the "Short" LRIMs, with 6 or 7 LRRs, also share the signal peptide, cysteine residue patterning, and coiled-coil sequence features of LRIM1 and APL1C. The "TM" LRIMs have a predicted C-terminal transmembrane region, and the "Coil-less" LRIMs exhibit the characteristic LRIM sequence signatures but lack the C-terminal coiled-coil domains. Conclusions The evolutionary plasticity of the LRIM LRR domains may provide templates for diverse recognition properties, while their coiled-coil domains could be involved in the formation

  15. Effects of the antimicrobial tylosin on the microbial community structure of an anaerobic sequencing batch reactor.

    Science.gov (United States)

    Shimada, Toshio; Li, Xu; Zilles, Julie L; Morgenroth, Eberhard; Raskin, Lutgarde

    2011-02-01

    The effects of the antimicrobial tylosin on a methanogenic microbial community were studied in a glucose-fed laboratory-scale anaerobic sequencing batch reactor (ASBR) exposed to stepwise increases of tylosin (0, 1.67, and 167 mg/L). The microbial community structure was determined using quantitative fluorescence in situ hybridization (FISH) and phylogenetic analyses of bacterial 16S ribosomal RNA (rRNA) gene clone libraries of biomass samples. During the periods without tylosin addition and with an influent tylosin concentration of 1.67 mg/L, 16S rRNA gene sequences related to Syntrophobacter were detected and the relative abundance of Methanosaeta species was high. During the highest tylosin dose of 167 mg/L, 16S rRNA gene sequences related to Syntrophobacter species were not detected and the relative abundance of Methanosaeta decreased considerably. Throughout the experimental period, Propionibacteriaceae and high GC Gram-positive bacteria were present, based on 16S rRNA gene sequences and FISH analyses, respectively. The accumulation of propionate and subsequent reactor failure after long-term exposure to tylosin are attributed to the direct inhibition of propionate-oxidizing syntrophic bacteria closely related to Syntrophobacter and the indirect inhibition of Methanosaeta by high propionate concentrations and low pH.

  16. Nearly identical bacteriophage structural gene sequences are widely distributed in both marine and freshwater environments.

    Science.gov (United States)

    Short, Cindy M; Suttle, Curtis A

    2005-01-01

    Primers were designed to amplify a 592-bp region within a conserved structural gene (g20) found in some cyanophages. The goal was to use this gene as a proxy to infer genetic richness in natural cyanophage communities and to determine if sequences were more similar in similar environments. Gene products were amplified from samples from the Gulf of Mexico, the Arctic, Southern, and Northeast and Southeast Pacific Oceans, an Arctic cyanobacterial mat, a catfish production pond, lakes in Canada and Germany, and a depth of ca. 3,246 m in the Chuckchi Sea. Amplicons were separated by denaturing gradient gel electrophoresis, and selected bands were sequenced. Phylogenetic analysis revealed four previously unknown groups of g20 clusters, two of which were entirely found in freshwater. Also, sequences with >99% identities were recovered from environments that differed greatly in temperature and salinity. For example, nearly identical sequences were recovered from the Gulf of Mexico, the Southern Pacific Ocean, an Arctic freshwater cyanobacterial mat, and Lake Constance, Germany. These results imply that closely related hosts and the viruses infecting them are distributed widely across environments or that horizontal gene exchange occurs among phage communities from very different environments. Moreover, the amplification of g20 products from deep in the cyanobacterium-sparse Chuckchi Sea suggests that this primer set targets bacteriophages other than those infecting cyanobacteria.

  17. Structural analysis of a repetitive protein sequence motif in strepsirrhine primate amelogenin.

    Directory of Open Access Journals (Sweden)

    Rodrigo S Lacruz

    Full Text Available Strepsirrhines are members of a primate suborder that has a distinctive set of features associated with the development of the dentition. Amelogenin (AMEL, the better known of the enamel matrix proteins, forms 90% of the secreted organic matrix during amelogenesis. Although AMEL has been sequenced in numerous mammalian lineages, the only reported strepsirrhine AMEL sequences are those of the ring-tailed lemur and galago, which contain a set of additional proline-rich tandem repeats absent in all other primates species analyzed to date, but present in some non-primate mammals. Here, we first determined that these repeats are present in AMEL from three additional lemur species and thus are likely to be widespread throughout this group. To evaluate the functional relevance of these repeats in strepsirrhines, we engineered a mutated murine amelogenin sequence containing a similar proline-rich sequence to that of Lemur catta. In the monomeric form, the MQP insertions had no influence on the secondary structure or refolding properties, whereas in the assembled form, the insertions increased the hydrodynamic radii. We speculate that increased AMEL nanosphere size may influence enamel formation in strepsirrhine primates.

  18. Viroids: from genotype to phenotype just relying on RNA sequence and structural motifs

    Directory of Open Access Journals (Sweden)

    Ricardo eFlores

    2012-06-01

    Full Text Available As a consequence of two unique physical properties, small size and circularity, viroid RNAs do not code for proteins and thus depend on RNA sequence/structural motifs for interacting with host proteins that mediate their invasion, replication, spread, and circumvention of defensive barriers. Viroid genomes fold up on themselves adopting collapsed secondary structures wherein stretches of nucleotides stabilized by Watson-Crick pairs are flanked by apparently unstructured loops. However, compelling data show that they are instead stabilized by alternative non-canonical pairs and that specific loops in the rod-like secondary structure, characteristic of Potato spindle tuber viroid and most other members of the family Pospiviroidae, are critical for replication and systemic trafficking. In contrast, rather than folding into a rod-like secondary structure, most members of the family Avsunvioidae adopt multibranched conformations occasionally stabilized by kissing loop interactions critical for viroid viability in vivo. Besides these most stable secondary structures, viroid RNAs alternatively adopt during replication transient metastable conformations containing elements of local higher-order structure, prominent among which are the hammerhead ribozymes catalyzing a key replicative step in the family Avsunvioidae, and certain conserved hairpins that also mediate replication steps in the family Pospiviroidae. Therefore, different RNA structures ⎯either global or local ⎯ determine different functions, thus highlighting the need for in-depth structural studies on viroid RNAs.

  19. Multilocus sequence subtyping and genetic structure of Cryptosporidium muris and Cryptosporidium andersoni.

    Science.gov (United States)

    Wang, Rongjun; Jian, Fuchun; Zhang, Longxian; Ning, Changshen; Liu, Aiqin; Zhao, Jinfeng; Feng, Yaoyu; Qi, Meng; Wang, Helei; Lv, Chaochao; Zhao, Guanghui; Xiao, Lihua

    2012-01-01

    In this study, nine C. muris and 43 C. andersoni isolates from various animals in China were subtyped by a multilocus sequence typing (MLST) tool. DNA sequence analyses showed the presence of 1-2 subtypes of C. muris and 2-6 subtypes of C. andersoni at each of the four loci (MS1, MS2, MS3, and MS16), nine of which represented new subtypes. Altogether, two C. muris and 10 C. andersoni MLST subtypes were detected. Linkage disequilibrium analysis indicated although the overall population structure of the two parasites was clonal, the Chinese C. andersoni in cattle has an epidemic structure. Three and two clusters were produced in the C. muris and C. andersoni populations by Structure 2.3.3 analysis, with Chinese C. muris and C. andersoni substructures differing from other countries. Thus, this study suggested the prevalence of C. andersoni in China is not attributed to the introduction of dairy cattle. More studies involving more genetic loci and systematic sampling are needed to better elucidate the population genetic structure of C. muris and C. andersoni in the world and the genetic basis for the difference in host specificity among the two most common gastric parasites.

  20. Genetic structure of Florida green turtle rookeries as indicated by mitochondrial DNA control region sequences

    Science.gov (United States)

    Shamblin, Brian M.; Bagley, Dean A.; Ehrhart, Llewellyn M.; Desjardin, Nicole A.; Martin, R. Erik; Hart, Kristen M.; Naro-Maciel, Eugenia; Rusenko, Kirt; Stiner, John C.; Sobel, Debra; Johnson, Chris; Wilmers, Thomas; Wright, Laura J.; Nairn, Campbell J.

    2014-01-01

    Green turtle (Chelonia mydas) nesting has increased dramatically in Florida over the past two decades, ranking the Florida nesting aggregation among the largest in the Greater Caribbean region. Individual beaches that comprise several hundred kilometers of Florida’s east coast and Keys support tens to thousands of nests annually. These beaches encompass natural to highly developed habitats, and the degree of demographic partitioning among rookeries was previously unresolved. We characterized the genetic structure of ten Florida rookeries from Cape Canaveral to the Dry Tortugas through analysis of 817 base pair mitochondrial DNA (mtDNA) control region sequences from 485 nesting turtles. Two common haplotypes, CM-A1.1 and CM-A3.1, accounted for 87 % of samples, and the haplotype frequencies were strongly partitioned by latitude along Florida’s Atlantic coast. Most genetic structure occurred between rookeries on either side of an apparent genetic break in the vicinity of the St. Lucie Inlet that separates Hutchinson Island and Jupiter Island, representing the finest scale at which mtDNA structure has been documented in marine turtle rookeries. Florida and Caribbean scale analyses of population structure support recognition of at least two management units: central eastern Florida and southern Florida. More thorough sampling and deeper sequencing are necessary to better characterize connectivity among Florida green turtle rookeries as well as between the Florida nesting aggregation and others in the Greater Caribbean region.

  1. Chromatin structure of repeating CTG/CAG and CGG/CCG sequences in human disease.

    Science.gov (United States)

    Wang, Yuh-Hwa

    2007-05-01

    In eukaryotic cells, chromatin structure organizes genomic DNA in a dynamic fashion, and results in regulation of many DNA metabolic processes. The CTG/CAG and CGG/CCG repeating sequences involved in several neuromuscular degenerative diseases display differential abilities for the binding of histone octamers. The effect of the repeating DNA on nucleosome assembly could be amplified as the number of repeats increases. Also, CpG methylation, and sequence interruptions within the triplet repeats exert an impact on the formation of nucleosomes along these repeating DNAs. The two most common triplet expansion human diseases, myotonic dystrophy 1 and fragile X syndrome, are caused by the expanded CTG/CAG and CGG/CCG repeats, respectively. In addition to the expanded repeats and CpG methylation, histone modifications, chromatin remodeling factors, and noncoding RNA have been shown to coordinate the chromatin structure at both myotonic dystrophy 1 and fragile X loci. Alterations in chromatin structure at these two loci can affect transcription of these disease-causing genes, leading to disease symptoms. These observations have brought a new appreciation that a full understanding of disease gene expression requires a knowledge of the structure of the chromatin domain within which the gene resides.

  2. One common structural feature of "words" in protein sequences and human texts.

    Science.gov (United States)

    Zemková, M; Trifonov, E N; Zahradník, D

    2014-01-01

    Frequently discussed analogy between genetic and human texts is explored by comparison of alternation of polar and non-polar amino-acid residues in proteins and alternation of consonants and vowels in human texts. In human languages, the usage of possible combinations of consonants and vowels is influenced by pronounceability of the combinations. Similarly, oligopeptide composition of proteins is influenced by requirements of protein folding and stability. One special type of structure often present in proteins is amphipathic α-helices in which polar and non-polar amino acids alternate with the period 3.5 residues, not unlike alternation of consonants and vowels. In this study, we evaluated the contribution made by amphipathic alternations to the protein sequence texts (20-24%). Their proportion is lower than respective values for alternating words in human texts (57-89%). The proteomes (full sets of proteins for selected organisms) were transformed into ranked sequences of n-grams (words of length n), including periodical amphipathic structures. Similarly, human texts were transformed into sequences of alternating consonants and vowels. Analysis of the vocabularies shows that in both types of texts (human languages and proteins) the alternating words are dominant or highly preferred, thus, strengthening the analogy between these two types of texts. The contribution of amphipathic words in the upper parts of the ranked lists for 10 analyzed proteomes varies between 58 and 74%. In human texts respective values range between 90 and 100%.

  3. Structural insights and ab initio sequencing within the DING proteins family

    Energy Technology Data Exchange (ETDEWEB)

    Elias, Mikael, E-mail: mikael.elias@weizmann.ac.il [Weizmann Institute of Science, Rehovot (Israel); Liebschner, Dorothee [CRM2, Nancy Université (France); Gotthard, Guillaume; Chabriere, Eric [AFMB, Université Aix-Marseille II (France)

    2011-01-01

    DING proteins constitute a recently discovered protein family that is ubiquitous in eukaryotes. The structural insights and the physiological involvements of these intriguing proteins are hereby deciphered. DING proteins constitute an intriguing family of phosphate-binding proteins that was identified in a wide range of organisms, from prokaryotes and archae to eukaryotes. Despite their seemingly ubiquitous occurrence in eukaryotes, their encoding genes are missing from sequenced genomes. Such a lack has considerably hampered functional studies. In humans, these proteins have been related to several diseases, like atherosclerosis, kidney stones, inflammation processes and HIV inhibition. The human phosphate binding protein is a human representative of the DING family that was serendipitously discovered from human plasma. An original approach was developed to determine ab initio the complete and exact sequence of this 38 kDa protein by utilizing mass spectrometry and X-ray data in tandem. Taking advantage of this first complete eukaryotic DING sequence, a immunohistochemistry study was undertaken to check the presence of DING proteins in various mice tissues, revealing that these proteins are widely expressed. Finally, the structure of a bacterial representative from Pseudomonas fluorescens was solved at sub-angstrom resolution, allowing the molecular mechanism of the phosphate binding in these high-affinity proteins to be elucidated.

  4. Evolutionary conservation of sequence and secondary structures inCRISPR repeats

    Energy Technology Data Exchange (ETDEWEB)

    Kunin, Victor; Sorek, Rotem; Hugenholtz, Philip

    2006-09-01

    Clustered Regularly Interspaced Palindromic Repeats (CRISPRs) are a novel class of direct repeats, separated by unique spacer sequences of similar length, that are present in {approx}40% of bacterial and all archaeal genomes analyzed to date. More than 40 gene families, called CRISPR-associated sequences (CAS), appear in conjunction with these repeats and are thought to be involved in the propagation and functioning of CRISPRs. It has been proposed that the CRISPR/CAS system samples, maintains a record of, and inactivates invasive DNA that the cell has encountered, and therefore constitutes a prokaryotic analog of an immune system. Here we analyze CRISPR repeats identified in 195 microbial genomes and show that they can be organized into multiple clusters based on sequence similarity. All individual repeats in any given cluster were inferred to form characteristic RNA secondary structure, ranging from non-existent to pronounced. Stable secondary structures included G:U base pairs and exhibited multiple compensatory base changes in the stem region, indicating evolutionary conservation and functional importance. We also show that the repeat-based classification corresponds to, and expands upon, a previously reported CAS gene-based classification including specific relationships between CRISPR and CAS subtypes.

  5. A replaceable liposomal aptamer for the ultrasensitive and rapid detection of biotin

    Science.gov (United States)

    Sung, Tzu-Cheng; Chen, Wen-Yih; Shah, Pramod; Chen, Chien-Sheng

    2016-02-01

    Biotin is an essential vitamin which plays an important role for maintaining normal physiological function. A rapid, sensitive, and simple method is necessary to monitor the biotin level. Here, we reported a replacement assay for the detection of biotin using a replaceable liposomal aptamer. Replacement assay is a competitive assay where a sample analyte replaces the labeled competitor of analyte out of its biorecognition element on a surface. It is user friendly and time-saving because of washing free. We used aptamer as a competitor, not a biorecognition element as tradition. To label aptamers, we used cholesterol-conjugated aptamers to tag signal-amplifying-liposomes. Without the need of conjugation procedure, aptamers can be easily incorporated into the surface of dye-encapsulating liposomes. Two aptamers as competitors of biotin, ST-21 and ST-21M with different affinities to streptavidin, were studied in parallel for the detection of biotin using replacement assays. ST-21 and ST-21M aptamers reached to limits of detection of 1.32 pg/80 μl and 0.47 pg/80 μl, respectively. The dynamic ranges of our assays using ST-21 and ST-21M aptamers were seven and four orders of magnitude, respectively. This assay can be completed in 20 minutes without washing steps. These results were overall better than previous reported assays.

  6. Combining aptamers and in silico interaction studies to decipher the function of hypothetical proteins

    DEFF Research Database (Denmark)

    Suravajhala, Prashanth; Burri, Harsha Vardhan Reddy; Heiskanen, Arto

    2014-01-01

    We present the potential role of aptamers in elucidating the function of hypothetical proteins, as well as the possibilities provided by bioinformatics for establishing a benchmark for aptamer-protein prediction methods. With these future perspectives, the role of hypothetical proteins as target...

  7. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

    Science.gov (United States)

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  8. Aptamers as a promising approach for the control of parasitic diseases

    Directory of Open Access Journals (Sweden)

    Juan David Ospina-Villa

    Full Text Available ABSTRACT Aptamers are short single-stranded RNA or DNA oligonucleotides that are capable of binding various biological targets with high affinity and specificity. Their identification initially relies on a molecular process named SELEX (Systematic Evolution of Ligands by EXponential enrichment that has been later modified in order to improve aptamer sensitivity, minimize duration and cost of the assay, as well as increase target types. Several biochemical modifications can help to enhance aptamer stability without affecting significantly target interaction. As a result, aptamers have generated a large interest as promising tools to compete with monoclonal antibodies for detection and inhibition of specific markers of human diseases. One aptamer-based drug is currently authorized and several others are being clinically evaluated. Despite advances in the knowledge of parasite biology and host-parasite interactions from "omics" data, protozoan parasites still affect millions of people around the world and there is an urgent need for drug target discovery and novel therapeutic concepts. In this context, aptamers represent promising tools for pathogen identification and control. Recent studies have reported the identification of "aptasensors" for parasite diagnosis, and "intramers" targeting intracellular proteins. Here we discuss various strategies that have been employed for intracellular expression of aptamers and expansion of their possible application, and propose that they may be suitable for the clinical use of aptamers in parasitic infections.

  9. Intracellular Expression of PAI-1 Specific Aptamers Alters Breast Cancer Cell Migration, Invasion and Angiogenesis.

    Science.gov (United States)

    Fortenberry, Yolanda M; Brandal, Stephanie M; Carpentier, Gilles; Hemani, Malvi; Pathak, Arvind P

    2016-01-01

    Plasminogen activator inhibitor-1 (PAI-1) is elevated in various cancers, where it has been shown to effect cell migration and invasion and angiogenesis. While, PAI-1 is a secreted protein, its intercellular levels are increased in cancer cells. Consequently, intracellular PAI-1 could contribute to cancer progression. While various small molecule inhibitors of PAI-1 are currently being investigated, none specifically target intracellular PAI-1. A class of inhibitors, termed aptamers, has been used effectively in several clinical applications. We previously generated RNA aptamers that target PAI-1 and demonstrated their ability to inhibit extracellular PAI-1. In the current study we explored the effect of these aptamers on intracellular PAI-1. We transiently transfected the PAI-1 specific aptamers into both MDA-MB-231 human breast cancer cells, and human umbilical vein endothelial cells (HUVECs) and studied their effects on cell migration, invasion and angiogenesis. Aptamer expressing MDA-MB-231 cells exhibited a decrease in cell migration and invasion. Additionally, intracellular PAI-1 and urokinase plasminogen activator (uPA) protein levels decreased, while the PAI-1/uPA complex increased. Moreover, a significant decrease in endothelial tube formation in HUVECs transfected with the aptamers was observed. In contrast, conditioned media from aptamer transfected MDA-MB-231 cells displayed a slight pro-angiogenic effect. Collectively, our study shows that expressing functional aptamers inside breast and endothelial cells is feasible and may exhibit therapeutic potential.

  10. DNA aptamer release from the DNA-SWNT hybrid by protein recognition.

    Science.gov (United States)

    Yoo, Chang-Hyuk; Jung, Seungwon; Bae, Jaehyun; Kim, Gunn; Ihm, Jisoon; Lee, Junghoon

    2016-02-14

    Here we show the formation of the complex between a DNA aptamer and a single-walled carbon nanotube (SWNT) and its reaction with its target protein. The aptamer, which is specifically bound with thrombin, the target protein in this study, easily wraps and disperses the SWNT by noncovalent π-π stacking.

  11. RNA aptamer-based electrochemical biosensor for selective and label-free analysis of dopamine

    DEFF Research Database (Denmark)

    Farjami, Elahe; Campos, Rui; Nielsen, Jesper Sejrup

    2013-01-01

    , including dopamine precursors and metabolites and other neurotransmitters (NT). Here we report an electrochemical RNA aptamer-based biosensor for analysis of dopamine in the presence of other NT. The biosensor exploits a specific binding of dopamine by the RNA aptamer, immobilized at a cysteamine...

  12. Intracellular Expression of PAI-1 Specific Aptamers Alters Breast Cancer Cell Migration, Invasion and Angiogenesis

    Science.gov (United States)

    Fortenberry, Yolanda M.; Brandal, Stephanie M.; Carpentier, Gilles; Hemani, Malvi; Pathak, Arvind P.

    2016-01-01

    Plasminogen activator inhibitor-1 (PAI-1) is elevated in various cancers, where it has been shown to effect cell migration and invasion and angiogenesis. While, PAI-1 is a secreted protein, its intercellular levels are increased in cancer cells. Consequently, intracellular PAI-1 could contribute to cancer progression. While various small molecule inhibitors of PAI-1 are currently being investigated, none specifically target intracellular PAI-1. A class of inhibitors, termed aptamers, has been used effectively in several clinical applications. We previously generated RNA aptamers that target PAI-1 and demonstrated their ability to inhibit extracellular PAI-1. In the current study we explored the effect of these aptamers on intracellular PAI-1. We transiently transfected the PAI-1 specific aptamers into both MDA-MB-231 human breast cancer cells, and human umbilical vein endothelial cells (HUVECs) and studied their effects on cell migration, invasion and angiogenesis. Aptamer expressing MDA-MB-231 cells exhibited a decrease in cell migration and invasion. Additionally, intracellular PAI-1 and urokinase plasminogen activator (uPA) protein levels d