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Sample records for aptamer molecular level

  1. Aptamers: molecular tools for analytical applications.

    Science.gov (United States)

    Mairal, Teresa; Ozalp, Veli Cengiz; Lozano Sánchez, Pablo; Mir, Mònica; Katakis, Ioanis; O'Sullivan, Ciara K

    2008-02-01

    Aptamers are artificial nucleic acid ligands, specifically generated against certain targets, such as amino acids, drugs, proteins or other molecules. In nature they exist as a nucleic acid based genetic regulatory element called a riboswitch. 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). Thanks to their unique characteristics and chemical structure, aptamers offer themselves as ideal candidates for use in analytical devices and techniques. Recent progress in the aptamer selection and incorporation of aptamers into molecular beacon structures will ensure the application of aptamers for functional and quantitative proteomics and high-throughput screening for drug discovery, as well as in various analytical applications. The properties of aptamers as well as recent developments in improved, time-efficient methods for their selection and stabilization are outlined. The use of these powerful molecular tools for analysis and the advantages they offer over existing affinity biocomponents are discussed. Finally the evolving use of aptamers in specific analytical applications such as chromatography, ELISA-type assays, biosensors and affinity PCR as well as current avenues of research and future perspectives conclude this review. PMID:17581746

  2. Current Progress of Aptamer-Based Molecular Imaging

    OpenAIRE

    Wang, Andrew Z.; Farokhzad, Omid C.

    2014-01-01

    Aptamers, single-stranded oligonucleotides, are an important class of molecular targeting ligand. Since their discovery, aptamers have been rapidly translated into clinical practice. They have been approved as therapeutics and molecular diagnostics. Aptamers also possess several properties that make them uniquely suited to molecular imaging. This review aims to provide an overview of aptamers’ advantages as targeting ligands and their application in molecular imaging.

  3. 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.

  4. DNA Aptamer Based Nanodrugs: Molecular Engineering for Efficiency.

    Science.gov (United States)

    Cansiz, Sena; Zhang, Liqin; Wu, Cuichen; Wu, Yuan; Teng, I-Ting; Hou, Weijia; Wang, Yanyue; Wan, Shuo; Cai, Ren; Jin, Chen; Liu, Qiaoling; Tan, Weihong

    2015-10-01

    In the past two decades, the study of cancer therapy has gradually advanced to the "nano" era. Numerous novel nanomaterials armed with unique physical properties have been introduced into biomedical research. At the same time, functional nucleic acid molecules, especially aptamers, have aroused broad attention from the biomedical community. Benefiting from the advancement of molecular engineering strategies, it is now feasible to combine the cancer-specific recognition capability of aptamers with various other special functions of nanomaterials to develop cancer-specific drugs at the nanoscale. Nanodrugs are now offering an unprecedented opportunity to achieve the goal of efficient targeted delivery as well as controlled release. This review highlights some achievements made in multiple aptamer-based nanodrug systems that have emerged in recent years, including studies in the infant stage of "proof-of-concept". PMID:26177853

  5. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites

    DEFF Research Database (Denmark)

    Dupont, Daniel Miotto; Thuesen, Cathrine K; Bøtkjær, Kenneth A;

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless......, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with...... therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to...

  6. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.

    Directory of Open Access Journals (Sweden)

    Daniel M Dupont

    Full Text Available Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126 with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA. We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A controlling uPA activities. One of the aptamers (upanap-126 binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12 binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

  7. 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. PMID:27591592

  8. Peptide aptamer identified by molecular docking targeting translationally controlled tumor protein in leukemia cells.

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    Kadioglu, Onat; Efferth, Thomas

    2016-08-01

    Bioinformatics screening and molecular docking analyses were utilized to select high affinity peptides targeting translationally controlled tumor protein (TCTP). Selected peptide aptamers were tested towards cancer cell lines with different levels of TCTP expression. One peptide (WGQWPYHC) revealed specific cytotoxicity according to the TCTP expression in tumor cells without affecting normal cells. Western blot analysis showed peptide-induced down-regulation of TCTP as primary target as well as of cell-cycle related downstream proteins (CDK2, CDK6, Cyclin D3) in MOLT-4 leukemia cells. "WGQWPYHC" deserves further analysis for targeted therapy of TCTP-expressing tumor cells. Graphical abstract Molecular docking on TCTP, cytotoxicity toward MOLT-4 leukemia cell line and downregulation of CDK2, CDK6, CyclinD3 and TCTP proteins. PMID:26972431

  9. Selecting Molecular Recognition. What Can Existing Aptamers Tell Us about Their Inherent Recognition Capabilities and Modes of Interaction?

    OpenAIRE

    Ralf Landgraf; Qian Zhang

    2012-01-01

    The use of nucleic acid derived aptamers has rapidly expanded since the introduction of SELEX in 1990. Nucleic acid aptamers have demonstrated their ability to target a broad range of molecules in ways that rival antibodies, but advances have been very uneven for different biochemical classes of targets, and clinical applications have been slow to emerge. What sets different aptamers apart from each other and from rivaling molecular recognition platforms, specifically proteins? What advantage...

  10. Selecting Molecular Recognition. What Can Existing Aptamers Tell Us about Their Inherent Recognition Capabilities and Modes of Interaction?

    Directory of Open Access Journals (Sweden)

    Ralf Landgraf

    2012-05-01

    Full Text Available The use of nucleic acid derived aptamers has rapidly expanded since the introduction of SELEX in 1990. Nucleic acid aptamers have demonstrated their ability to target a broad range of molecules in ways that rival antibodies, but advances have been very uneven for different biochemical classes of targets, and clinical applications have been slow to emerge. What sets different aptamers apart from each other and from rivaling molecular recognition platforms, specifically proteins? What advantages do aptamers as a reagent class offer, and how do the chemical properties and selection procedures of aptamers influence their function? Do the building blocks of nucleic acid aptamers dictate inherent limitations in the nature of molecular targets, and do existing aptamers give us insight in how these challenges might be overcome? This review is written as an introduction for potential endusers of aptamer technology who are evaluating the advantages of aptamers as a versatile, affordable, yet highly expandable platform to target a broad range of biological processes or interactions.

  11. Molecular Selection, Modification and Development of Therapeutic Oligonucleotide Aptamers

    OpenAIRE

    Yuanyuan Yu; Chao Liang; Quanxia Lv; Defang Li; Xuegong Xu; Baoqin Liu; Aiping Lu; Ge Zhang

    2016-01-01

    Monoclonal antibodies are the dominant agents used in inhibition of biological target molecules for disease therapeutics, but there are concerns of immunogenicity, production, cost and stability. Oligonucleotide aptamers have comparable affinity and specificity to targets with monoclonal antibodies whilst they have minimal immunogenicity, high production, low cost and high stability, thus are promising inhibitors to rival antibodies for disease therapy. In this review, we will compare the det...

  12. Rapid determination of total hardness in water using fluorescent molecular aptamer beacon

    Energy Technology Data Exchange (ETDEWEB)

    Lerga, T. Mairal [Nanobiotechnology and Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira I Virgili (Spain); O' Sullivan, Ciara K. [Nanobiotechnology and Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira I Virgili (Spain); Institucio Catalana de Recerca i Estudis Avancats, Passeig Lluis Companys 23, 08010 Barcelona (Spain)], E-mail: ciara.osullivan@urv.net

    2008-03-03

    A double-labelled synthetic oligonucleotide is used as a fluorescent molecular aptamer beacon for the reagentless determination of total hardness in tap and bottled waters. Modified thrombin binding aptamer (5'-NH-C3-GGTTGGTGTGGTTGG-C3-SH-3') carrying 6-carboxyfluorescein (FAM) and 7-amino-4-methyl-coumarin labels at 5' and 3', respectively, was used for the simultaneous combined measurement of Mg{sup 2+} and Ca{sup 2+} cations. Interference from the K{sup +} cation is eliminated via selective tuning of the assay conditions, increasing the temperature beyond the melting point of the potassium-stabilised quadruplex facilitating its liberation from the quadruplex, whilst maintaining the integrity of the magnesium/calcium-stabilised structure. No interference from other cations found in tap or bottled water was observed. The detection limit of the aptamer beacon is 0.04 mmol L{sup -1}, with a dynamic linear range of 0-0.5 {mu}M and is very reproducible, with an R.S.D. = 8%, n = 3. The fluorescent molecular beacon is applied to the determination of total hardness in tap and bottled waters and its' performance compared to that of the standard method of complexiometric titration and atomic absorption spectroscopy, with an excellent correlation observed. Further work is focused on the immobilization of the aptamer for the development of a re-usable fluorescent/electrochemical aptasensor, for the determination of water hardness.

  13. Rapid determination of total hardness in water using fluorescent molecular aptamer beacon

    International Nuclear Information System (INIS)

    A double-labelled synthetic oligonucleotide is used as a fluorescent molecular aptamer beacon for the reagentless determination of total hardness in tap and bottled waters. Modified thrombin binding aptamer (5'-NH-C3-GGTTGGTGTGGTTGG-C3-SH-3') carrying 6-carboxyfluorescein (FAM) and 7-amino-4-methyl-coumarin labels at 5' and 3', respectively, was used for the simultaneous combined measurement of Mg2+ and Ca2+ cations. Interference from the K+ cation is eliminated via selective tuning of the assay conditions, increasing the temperature beyond the melting point of the potassium-stabilised quadruplex facilitating its liberation from the quadruplex, whilst maintaining the integrity of the magnesium/calcium-stabilised structure. No interference from other cations found in tap or bottled water was observed. The detection limit of the aptamer beacon is 0.04 mmol L-1, with a dynamic linear range of 0-0.5 μM and is very reproducible, with an R.S.D. = 8%, n = 3. The fluorescent molecular beacon is applied to the determination of total hardness in tap and bottled waters and its' performance compared to that of the standard method of complexiometric titration and atomic absorption spectroscopy, with an excellent correlation observed. Further work is focused on the immobilization of the aptamer for the development of a re-usable fluorescent/electrochemical aptasensor, for the determination of water hardness

  14. Analyzing models for interactions of aptamers to proteins

    Science.gov (United States)

    Silva, Dilson; Missailidis, Sotiris

    2014-10-01

    We have devised an experimental and theoretical model, based on fluorescent spectroscopy and molecular modelling, to describe the interaction of aptamer (selected against various protein targets) with proteins and albumins in particular. This model, described in this work, has allowed us to decipher the nature of the interactions between aptamers and albumins, the binding site of the aptamers to albumins, the potential role of primer binding to the albumin and expand to the ability of albumin to carry aptamers in the bloodstream, providing data to better understand the level of free aptamer for target binding. We are presenting the study of a variety of aptamers, including those against the MUC1 tumour marker, heparanase and human kallikrein 6 with bovine and human serum albumins and the effect these interactions may have on the bioavailability of the aptamer for target-specific binding and therapeutic activity.

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

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    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

  16. Aptamer therapeutics: A review of current practice

    International Nuclear Information System (INIS)

    Full text: The development of nuclease resistant oligonucleotide agents known as aptamers, offers an alternative to antibodies as targeting, diagnostic and delivery agents. The production technique of specific receptor binding molecules based on defined nucleic acid sequences is known as systematic evolution of ligands by exponential enrichment (SELEX). Using this technique, aptamers can be produced rapidly and with high homogeneity. Furthermore, they are stable over long term storage at ambient room temperatures. A monomeric aptamer is small in size, with a molecular weight as low as 5 to 10 kDa. However, the aptamer molecule may be used as a building block for custom designed targeting agents, offering several advantages. Aptamers have been found to bind their targets with high specificity and with dissociation constants in the subnanomolar or picomolar range. The first pharmaceutical aptamer formulation, Macugen (pegaptanib sodium injection) was approved in the United States in December of 2004. This is an anti-VEGF aptamer formulation used for the treatment of Neovascular agerelated macular degeneration. Other possibilities in cardiovascular, neurodegenerative and tropical medicine are apparent. As tumour targeting agents, aptamers penetrate tissues readily, reach peak levels quickly and clear from the body rapidly, thus having properties of low toxicity and immunoreactivity. Work with radiolabelled aptamers is limited to pre-clinical studies, but the body of evidence is steadily growing and aptamers are emerging as valuable clinical products for diagnostic imaging and therapy. Peptide coupling reactions between amino and carboxylic groups offer the possibility of labelling the aptamers with a number of chelators that, coupled with appropriate radionuclides, would generate novel targeted radiopharmaceuticals for the diagnosis and therapy of disease. The unparalleled combinatorial chemical diversity, small size and modification ability of aptamers is expected to

  17. High-resolution single-molecule recognition imaging of the molecular details of ricin-aptamer interaction

    Science.gov (United States)

    The molecular details of DNA aptamer-ricin interactions were investigated. The toxic protein ricin molecules were immobilized on Au(111) surface using N-hydroxysuccinimide (NHS) ester to specifically react with lysine residues located on the ricin B chains. A single ricin molecule was visualized in ...

  18. Generating Cell Targeting Aptamers for Nanotheranostics Using Cell-SELEX

    Science.gov (United States)

    Lyu, Yifan; Chen, Guang; Shangguan, Dihua; Zhang, Liqin; Wan, Shuo; Wu, Yuan; Zhang, Hui; Duan, Lian; Liu, Chao; You, Mingxu; Wang, Jie; Tan, Weihong

    2016-01-01

    Detecting and understanding changes in cell conditions on the molecular level is of great importance for the accurate diagnosis and timely therapy of diseases. Cell-based SELEX (Systematic Evolution of Ligands by EXponential enrichment), a foundational technology used to generate highly-specific, cell-targeting aptamers, has been increasingly employed in studies of molecular medicine, including biomarker discovery and early diagnosis/targeting therapy of cancer. In this review, we begin with a mechanical description of the cell-SELEX process, covering aptamer selection, identification and identification, and aptamer characterization; following this introduction is a comprehensive discussion of the potential for aptamers as targeting moieties in the construction of various nanotheranostics. Challenges and prospects for cell-SELEX and aptamer-based nanotheranostic are also discussed. PMID:27375791

  19. Nucleolin-aptamer therapy in retinoblastoma: molecular changes and mass spectrometry-based imaging.

    Science.gov (United States)

    Subramanian, Nithya; Srimany, Amitava; Kanwar, Jagat R; Kanwar, Rupinder K; Akilandeswari, Balachandran; Rishi, Pukhraj; Khetan, Vikas; Vasudevan, Madavan; Pradeep, Thalappil; Krishnakumar, Subramanian

    2016-01-01

    Retinoblastoma (RB) is an intraocular childhood tumor which, if left untreated, leads to blindness and mortality. Nucleolin (NCL) protein which is differentially expressed on the tumor cell surface, binds ligands and regulates carcinogenesis and angiogenesis. We found that NCL is over expressed in RB tumor tissues and cell lines compared to normal retina. We studied the effect of nucleolin-aptamer (NCL-APT) to reduce proliferation in RB tumor cells. Aptamer treatment on the RB cell lines (Y79 and WERI-Rb1) led to significant inhibition of cell proliferation. Locked nucleic acid (LNA) modified NCL-APT administered subcutaneously (s.c.) near tumor or intraperitoneally (i.p.) in Y79 xenografted nude mice resulted in 26 and 65% of tumor growth inhibition, respectively. Downregulation of inhibitor of apoptosis proteins, tumor miRNA-18a, altered serum cytokines, and serum miRNA-18a levels were observed upon NCL-APT treatment. Desorption electrospray ionization mass spectrometry (DESI MS)-based imaging of cell lines and tumor tissues revealed changes in phosphatidylcholines levels upon treatment. Thus, our study provides proof of concept illustrating NCL-APT-based targeted therapeutic strategy and use of DESI MS-based lipid imaging in monitoring therapeutic responses in RB. PMID:27574784

  20. Microfluidic approaches to rapid and efficient aptamer selection

    OpenAIRE

    Lin, Hui; Zhang, Weiting; Jia, Shasha; Guan, Zhichao; Yang, Chaoyong James; Zhu, Zhi

    2014-01-01

    With their advantages as molecular recognition elements, aptamers have been extensively studied and used for bioanalytical and biomedical applications. However, the process of enrichment and screening of aptamers remains a bottleneck for aptamer development. Recently, microfluidic methods have been increasingly used for rapid and efficient aptamer selection, showing their remarkable advantages over conventional methods. This review briefly introduces aptamers and their advantages. The convent...

  1. Aptamers and aptamer targeted delivery

    OpenAIRE

    Yan, Amy C.; Levy, Matthew

    2009-01-01

    When aptamers first emerged almost two decades ago, most were RNA species that bound and tagged or inhibited simple target ligands. Very soon after, the ‘selectionologists’ developing aptamer technology quickly realized more potential for the aptamer. In recent years, advances in aptamer techniques have enabled the use of aptamers as small molecule inhibitors, diagnostic tools and even therapeutics. Aptamers are now being employed in novel applications. We review, herein, some of the recent a...

  2. Radiolabelled aptamers for tumour imaging and therapy

    International Nuclear Information System (INIS)

    , where four aptamers were attached to the four arms of either DOTA or carboxy-porphyrin. The four complexes were labelled with Tc-99m and tested for their efficacy as tumour imaging agents. All four complexes demonstrated specificity for the tumour, due to their MUC1 specificity, at various levels. Biodistribution studies were carried out in mice with MCF7 xenografts. The monomeric aptamer complexes had rapid renal clearance from the system, due to their small size (MW of 8,000 Da). More than 90% of the aptamer based radiopharmaceutical was cleared from the system within the first 15 minutes. To increase retention time, additional constructs based on the design of a tetra-aptamer complex were prepared. A core chelator, such as DOTA and carboxy-porphyrin has been used as a skeleton for the building of multiaptamer constructs aiming to increase the molecular weight of the complex and potentially its stability of binding due to interactions with more than one MUC1 molecules at the surface of the tumour cell. The increase of the MW to 32,000 Da allowed increased retention times in the system, without compromising the exceptional tumour penetration exhibited by all the aptamer based constructs under study. The development of aptamers as small building blocks for targeting agents offers several advantages. These molecules penetrate tumour more readily than whole antibodies, reach peak levels in the tumour more rapidly and clear from the body faster, thereby reducing toxicity to healthy tissues. Our strategy is to manipulate the molecular weight of the construct utilising previously devised methodologies to achieve various polymeric aptamer complexes in order to achieve the optimum balance between the low immunogenicity and excellent tumour penetration. In this way we aim to achieve a balance against the rapid renal clearance that leads to premature elimination of the complex from the system and adequate tumour uptake for diagnostic imaging and targeted therapy. We intend to

  3. Generating Aptamers by Cell-SELEX for Applications in Molecular Medicine

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    Huixia Liu

    2012-03-01

    Full Text Available Aptamers are single-stranded oligonucleotides of DNA or RNA that bind to target molecules with high affinity and specificity. Typically, aptamers are generated by an iterative selection process, called systematic evolution of ligands by exponential enrichment (SELEX. Recent advancements in SELEX technology have extended aptamer selection from comparatively simple mixtures of purified proteins to whole living cells, and now cell-based SELEX (or cell-SELEX can isolate aptamers that bind to specific target cells. Combined with nanotechnology, microchips, microfluidic devices, RNAi and other advanced technologies, cell-SELEX represents an integrated platform providing ultrasensitive and highly specific tools for clinical medicine. In this review, we describe the recent progress made in the application of cell-SELEX for diagnosis, therapy and biomarker discovery.

  4. Unfolding mechanism of thrombin-binding aptamer revealed by molecular dynamics simulation and Markov State Model

    Science.gov (United States)

    Zeng, Xiaojun; Zhang, Liyun; Xiao, Xiuchan; Jiang, Yuanyuan; Guo, Yanzhi; Yu, Xinyan; Pu, Xuemei; Li, Menglong

    2016-04-01

    Thrombin-binding aptamer (TBA) with the sequence 5‧GGTTGGTGTGGTTGG3‧ could fold into G-quadruplex, which correlates with functionally important genomic regionsis. However, unfolding mechanism involved in the structural stability of G-quadruplex has not been satisfactorily elucidated on experiments so far. Herein, we studied the unfolding pathway of TBA by a combination of molecular dynamics simulation (MD) and Markov State Model (MSM). Our results revealed that the unfolding of TBA is not a simple two-state process but proceeds along multiple pathways with multistate intermediates. One high flux confirms some observations from NMR experiment. Another high flux exhibits a different and simpler unfolding pathway with less intermediates. Two important intermediate states were identified. One is similar to the G-triplex reported in the folding of G-quadruplex, but lack of H-bonding between guanines in the upper plane. More importantly, another intermediate state acting as a connector to link the folding region and the unfolding one, was the first time identified, which exhibits higher population and stability than the G-triplex-like intermediate. These results will provide valuable information for extending our understanding the folding landscape of G-quadruplex formation.

  5. Combinatorial selection of aptamers: new radioligands for in vivo molecular imaging

    International Nuclear Information System (INIS)

    Aptamers are oligonucleotide structures selected for their capacity to bind to a desired target. The first part of this work focuses on the selection of aptamers directed against the oncogenic form of the tyrosine kinase receptor Ret (RetC634Y). We compared different selection protocols: i) selection against the purified RetC634Y recombinant protein, ii) selection against whole living cells which express RetC634Y and iii) a crossover selection alternating between cells and recombinant protein. One aptamer, D4, was found to be able to inhibit Ret and to reverse the cell phenotype induced by the activation of the receptor. Then, we developed the in vivo use of the selected aptamers. Finally, we used the whole living cells selection protocol to develop aptamers against HLA-G. This protein is characterised by its function in immuno tolerance. Taken together, these studies should pave the way for the in vivo use of aptamers as new therapeutic and diagnostic agents for in vivo PET imaging. (author)

  6. Two-photon graphene oxide/aptamer nanosensing conjugate for in vitro or in vivo molecular probing.

    Science.gov (United States)

    Yi, Mei; Yang, Sheng; Peng, Zanying; Liu, Changhui; Li, Jishan; Zhong, Wenwan; Yang, Ronghua; Tan, Weihong

    2014-04-01

    Two-photon excitation (TPE) with near-infrared (NIR) photons as the excitation source have the unique properties of lower tissue autofluorescence and self-absorption, reduced photodamage and photobleaching, higher spatial resolution, and deeper penetration depth (>500 μm). Carbon nanomaterials, for example, graphene oxide (GO), have the advantages of good biocompatibility, efficient transporters into cells, protecting the carried DNA or peptides from enzymatic cleavage, and super fluorescence quenching efficiency. By combination of the nanostructured carbon materials with the TPE technique, herein we have designed an aptamer-two-photon dye (TPdye)/GO TPE fluorescent nanosensing conjugate for molecular probing in biological fluids, living cells, and zebrafish. This approach takes advantage of the exceptional quenching capability of GO for the proximate TP dyes and the higher affinity of single-stranded DNA on GO than the aptamer-target complex. Successful in vitro and in vivo detection of ATP was demonstrated with this sensing strategy. Our results reveal that the GO/Aptamer-TPdye system not only is a robust, sensitive, and selective sensor for quantitative detection of ATP in the complex biological environment but also can be efficiently delivered into live cells or tissues and act as a "signal-on" in vivo sensor for specific, high-contrast imaging of target biomolecules. Our design provides a methodology model scheme for development of future carbon nanomaterial-based two-photon fluorescent probes for in vitro or in vivo determination of biological or biologically relevant species. PMID:24592855

  7. Molecular and Functional Characterization of ssDNA Aptamers that Specifically Bind Leishmania infantum PABP.

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    Natalia Guerra-Pérez

    Full Text Available A poly (A-binding protein from Leishmania infantum (LiPABP has been recently cloned and characterized in our laboratory. Although this protein shows a very high homology with PABPs from other eukaryotic organisms including mammals and other parasites, exist divergences along the sequence that convert them in potential diagnostic markers and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania proteins is essential for the progress of this kind of study.We have selected a ssDNA aptamer population against a recombinant 6xHIS-LiPABP protein (rLiPABP that is able to recognize the target with a low Kd. Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11 that significantly bound to PABP with higher affinity than the naïve population. These aptamers were analyzed by ELONA and slot blot to establish affinity and specificity for rLiPABP. Results demonstrated that the three aptamers have high affinity and specificity for the target and that they are able to detect an endogenous LiPABP (eLiPABP protein amount corresponding to 2500 L. infantum promastigotes in a significant manner. The functional analysis of the aptamers also revealed that ApPABP#11 disrupts the binding of both Myc-LiPABP and eLiPABP to poly (A in vitro. On the other hand, these aptamers are able to bind and purify LiPABP from complex mixes.Results presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity. At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be analyzed.

  8. Aptamer-modified magnetic nanoprobe for molecular MR imaging of VEGFR2 on angiogenic vasculature

    Science.gov (United States)

    Kim, Bongjune; Yang, Jaemoon; Hwang, Myeonghwan; Choi, Jihye; Kim, Hyun-Ouk; Jang, Eunji; Lee, Jung Hwan; Ryu, Sung-Ho; Suh, Jin-Suck; Huh, Yong-Min; Haam, Seungjoo

    2013-09-01

    Nucleic acid-based aptamers have been developed for the specific delivery of diagnostic nanoprobes. Here, we introduce a new class of smart imaging nanoprobe, which is based on hybridization of a magnetic nanocrystal with a specific aptamer for specific detection of the angiogenic vasculature of glioblastoma via magnetic resonance (MR) imaging. The magnetic nanocrystal imaging core was synthesized using the thermal decomposition method and enveloped by carboxyl polysorbate 80 for water solubilization and conjugation of the targeting moiety. Subsequently, the surface of the carboxylated magnetic nanocrystal was modified with amine-functionalized aptamers that specifically bind to the vascular growth factor receptor 2 (VEGFR2) that is overexpressed on angiogenic vessels. To assess the targeted imaging potential of the aptamer-conjugated magnetic nanocrystal for VEGFR2 markers, the magnetic properties and MR imaging sensitivity were investigated using the orthotopic glioblastoma mouse model. In in vivo tests, the aptamer-conjugated magnetic nanocrystal effectively targeted VEGFR2 and demonstrated excellent MR imaging sensitivity with no cytotoxicity.

  9. 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.

  10. Development of RNA aptamers as molecular probes for HER2+ breast cancer study using cell-SELEX

    Directory of Open Access Journals (Sweden)

    Seyedeh Alia Moosavian

    2015-06-01

    Full Text Available Objective(s: Development of molecules that specifically recognize cancer cells is one of the major areas in cancer research. Human epidermal growth factor receptor 2 (HER2 is specifically expressed on the surface of breast cancer cells. HER2 is associated with an aggressive phenotype and poor prognosis. In this study we aimed to isolate RNA aptamers that specifically bind to HER2 overexpressing TUBO cell line. Materials and Methods: Panel of aptamers was selected using cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX. Results: Binding studies showed that selected aptamers can identify TUBO cell line with high affinity and selectivity. Our preliminary investigation of the target of aptamers suggested that aptamers bind with HER2 proteins on the surface of TUBO cells. Conclusion: We believe the selected aptamers could be useful ligands for targeted breast cancer therapy.

  11. An investigation on the interaction modes of a single-strand DNA aptamer and RBP4 protein: a molecular dynamic simulations approach.

    Science.gov (United States)

    Torabi, Raheleh; Bagherzadeh, Kowsar; Ghourchian, Hedayatollah; Amanlou, Massoud

    2016-09-14

    Type two diabetes is one of the primary health issues threatening public well-being worldwide. One of the pre-diagnosis biomarkers of this disease, retinol binding protein 4 (RBP4), has been demonstrated to be detected with a 76-mer ssDNA aptamer instead of conventional antibodies. However, there is no structural information on the RBP4 binding aptamer (RBA) and the mechanism of its binding to RBP4 still remains unexplored. The objective of the present study is to achieve a better understanding of specific binding interactions of the target protein (RBP4) and RBA, employing Molecular Dynamics simulations (MDs) to provide detailed information on fluctuations, conformational changes, critical bases and effective forces to develop regulated aptamers to be employed in designing new aptamers for many useful recognition applications. RBA was designed according to its reported base pair sequence and secondary structure. The HADDOCK on line docking program was used to predict a suitable RBP4-RBA mode of interaction to start MDs with. MDs methodology was used to analyze the final complex stability and detect interacting residues. Eventually, we conclude that single strand located bases are the key components that conduct the intercalation phenomenon with big targets rather than those involving loops and folded motifs, to encompass targets and probably inhibit their activity. Also, UV-visible, circular dichroism and fluorescence spectroscopy measurements confirmed the interactions between RBA and RBP4 and RBP4-RBA complex formation. PMID:27511589

  12. Aptamer Microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Angel-Syrett, Heather; Collett, Jim; Ellington, Andrew D.

    2009-01-02

    In vitro selection can yield specific, high-affinity aptamers. We and others have devised methods for the automated selection of aptamers, and have begun to use these reagents for the construction of arrays. Arrayed aptamers have proven to be almost as sensitive as their solution phase counterparts, and when ganged together can provide both specific and general diagnostic signals for proteins and other analytes. We describe here technical details regarding the production and processing of aptamer microarrays, including blocking, washing, drying, and scanning. We will also discuss the challenges involved in developing standardized and reproducible methods for binding and quantitating protein targets. While signals from fluorescent analytes or sandwiches are typically captured, it has proven possible for immobilized aptamers to be uniquely coupled to amplification methods not available to protein reagents, thus allowing for protein-binding signals to be greatly amplified. Into the future, many of the biosensor methods described in this book can potentially be adapted to array formats, thus further expanding the utility of and applications for aptamer arrays.

  13. Investigating the malleability of RNA aptamers

    Energy Technology Data Exchange (ETDEWEB)

    Ilgu, Muslum; Wang, Tianjiao; Lamm, Monica H.; Nilsen-Hamilton, Marit

    2013-03-25

    Aptamers are short, single-stranded nucleic acids with structures that frequently change upon ligand binding and are sensitive to the ionic environment. To achieve facile application of aptamers in controlling cellular activities, a better understanding is needed of aptamer ligand binding parameters, structures, intramolecular mobilities and how these structures adapt to different ionic environments with consequent effects on their ligand binding characteristics.The paper discusses the integration of biochemical analysis with NMR spectroscopy and computational modeling to explore the relation between ligand binding and structural malleability of some well-studied aptamers. Several methods for determining aptamer binding affinity and specificity are discussed, including isothermal titration calorimetry, steady state fluorescence of 2-aminopurine substituted aptamers, and dye displacement assays. Also considered are aspects of molecular dynamics simulations specific to aptamers including adding ions and simulating aptamer structure in the absence of ligand when NMR spectroscopy or X-ray crystallography structures of the unoccupied aptamer are not available. We focus specifically on RNA aptamers that bind small molecule ligands as would be applied in sensors or integrated into riboswitches such as to measure the products of metabolic activity.

  14. Kinetic and Stoichiometric Characterisation of Streptavidin-Binding Aptamers

    NARCIS (Netherlands)

    Ruigrok, V.J.B.; Duijn, van E.; Barendregt, A.; Dyer, K.; Tainer, J.A.; Stoltenburg, R.; Strehlitz, B.; Levisson, M.; Smidt, H.; Oost, van der J.

    2012-01-01

    Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer-target interactions is available. To this end, streptavidin-binding aptamers were isolated

  15. Development of RNA aptamers as molecular probes for HER2+ breast cancer study using cell-SELEX

    OpenAIRE

    Seyedeh Alia Moosavian; Mahmoud Reza Jaafari; Seyed Mohammad Taghdisi; Fatemeh Mosaffa; Ali Badiee; Khalil Abnous

    2015-01-01

    Objective(s): Development of molecules that specifically recognize cancer cells is one of the major areas in cancer research. Human epidermal growth factor receptor 2 (HER2) is specifically expressed on the surface of breast cancer cells. HER2 is associated with an aggressive phenotype and poor prognosis. In this study we aimed to isolate RNA aptamers that specifically bind to HER2 overexpressing TUBO cell line. Materials and Methods: Panel of aptamers was selected using cell-based systematic...

  16. Molecular Mechanism for Inhibition of G Protein-Coupled Receptor Kinase 2 by a Selective RNA Aptamer

    Energy Technology Data Exchange (ETDEWEB)

    Tesmer, Valerie M.; Lennarz, Sabine; Mayer, Günter; Tesmer, John J.G. (Bonn); (Michigan)

    2012-08-31

    Cardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic {alpha}F-{alpha}G loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high-affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase.

  17. Study of the Molecular Recognition of Aptamers Selected through Ovarian Cancer Cell-SELEX

    OpenAIRE

    Dimitri Van Simaeys; Dalia López-Colón; Kwame Sefah; Rebecca Sutphen; Elizabeth Jimenez; Weihong Tan

    2010-01-01

    BACKGROUND: Ovarian cancer is the most lethal gynecological malignancy, and the ovarian clear cell carcinoma subtype (OCCA) demonstrates a particularly poor response to standard treatment. Improvements in ovarian cancer outcomes, especially for OCCA, could be expected from a clearer understanding of the molecular pathology that might guide strategies for earlier diagnosis and more effective treatment. METHODOLOGY/PRINCIPAL FINDINGS: Cell-SELEX technology was employed to develop new molecular ...

  18. Screening and Identification of ssDNA Aptamer for Human GP73

    Directory of Open Access Journals (Sweden)

    Jingchun Du

    2015-01-01

    Full Text Available As one tumor marker of HCC, Golgi Protein 73 (GP73 is given more promise in the early diagnosis of HCC, and aptamers have been developed to compete with antibodies as biorecognition probes in different detection system. In this study, we utilized GP73 to screen specific ssDNA aptamers by SELEX technique. First, GP73 proteins were expressed and purified by prokaryotic expression system and Nickle ion affinity chromatography, respectively. At the same time, the immunogenicity of purified GP73 was confirmed by Western blotting. The enriched ssDNA library with high binding capacity for GP73 was obtained after ten rounds of SELEX. Then, thirty ssDNA aptamers were sequenced, in which two ssDNA aptamers with identical DNA sequence were confirmed, based on the alignment results, and designated as A10-2. Furthermore, the specific antibody could block the binding of A10-2 to GP73, and the specific binding of A10-2 to GP73 was also supported by the observation that several tumor cell lines exhibited variable expression level of GP73. Significantly, the identified aptamer A10-2 could distinguish normal and cancerous liver tissues. So, our results indicate that the aptamer A10-2 might be developed into one molecular probe to detect HCC from normal liver specimens.

  19. Interactions of aptamers with sera albumins

    Science.gov (United States)

    Cortez, Célia Martins; Silva, Dilson; Silva, Camila M. C.; Missailidis, Sotiris

    2012-09-01

    The interactions of two short aptamers to human and bovine serum albumins were studied by fluorescence spectroscopic techniques. Intrinsic fluorescence of BSA and HSA were measured by selectively exciting their tryptophan residues. Gradual quenching was observed by titration of both proteins with aptamers. Aptamers are oligonucleic acid or peptide molecules that bind a specific target and can be used for both biotechnological and clinical purposes, since they present molecular recognition properties like that commonly found in antibodies. Two aptamers previously selected against the MUC1 tumour marker were used in this study, one selected for the protein core and one for the glycosylated MUC1. Stern-Volmer graphs were plotted and quenching constants were estimated. Plots obtained from experiments carried out at 25 °C and 37 °C showed the quenching of fluorescence of by aptamers to be a collisional phenomenon. Stern-Volmer constants estimated for HSA quenched by aptamer A were 1.68 × 105 (±5 × 103) M-1 at 37 °C, and 1.37 × 105 (±103) M-1 at 25 °C; and quenched by aptamer B were 1.67 × 105 (±5 × 103) M-1 at 37 °C, and 1.32 × 105 (±103) M-1 at 25 °C. Results suggest that the primary binding site for aptamers on albumin is close to tryptophan residues in sub domain IIA.

  20. Applications of Aptamers in Targeted Imaging: State of the Art

    OpenAIRE

    Dougherty, Casey A.; Cai, Weibo; Hong, Hao

    2015-01-01

    Aptamers are single-stranded oligonucleotides with high affinity and specificity to the target molecules or cells, thus they can serve as an important category of molecular targeting ligand. Since their discove1y, aptamers have been rapidly translated into clinical practice. The strong target affinity/selectivity, cost-effectivity, chemical versatility and safety of aptamers are superior to traditional peptides- or proteins-based ligands which make them unique choices for molecular imaging. T...

  1. Designing anti-influenza aptamers: novel quantitative structure activity relationship approach gives insights into aptamer-virus interaction.

    Directory of Open Access Journals (Sweden)

    Boaz Musafia

    Full Text Available This study describes the development of aptamers as a therapy against influenza virus infection. Aptamers are oligonucleotides (like ssDNA or RNA that are capable of binding to a variety of molecular targets with high affinity and specificity. We have studied the ssDNA aptamer BV02, which was designed to inhibit influenza infection by targeting the hemagglutinin viral protein, a protein that facilitates the first stage of the virus' infection. While testing other aptamers and during lead optimization, we realized that the dominant characteristics that determine the aptamer's binding to the influenza virus may not necessarily be sequence-specific, as with other known aptamers, but rather depend on general 2D structural motifs. We adopted QSAR (quantitative structure activity relationship tool and developed computational algorithm that correlate six calculated structural and physicochemical properties to the aptamers' binding affinity to the virus. The QSAR study provided us with a predictive tool of the binding potential of an aptamer to the influenza virus. The correlation between the calculated and actual binding was R2 = 0.702 for the training set, and R2 = 0.66 for the independent test set. Moreover, in the test set the model's sensitivity was 89%, and the specificity was 87%, in selecting aptamers with enhanced viral binding. The most important properties that positively correlated with the aptamer's binding were the aptamer length, 2D-loops and repeating sequences of C nucleotides. Based on the structure-activity study, we have managed to produce aptamers having viral affinity that was more than 20 times higher than that of the original BV02 aptamer. Further testing of influenza infection in cell culture and animal models yielded aptamers with 10 to 15 times greater anti-viral activity than the BV02 aptamer. Our insights concerning the mechanism of action and the structural and physicochemical properties that govern the interaction

  2. Hybridization-based aptamer labeling using complementary oligonucleotide platform for PET and optical imaging.

    Science.gov (United States)

    Park, Jun Young; Lee, Tae Sup; Song, In Ho; Cho, Ye Lim; Chae, Ju Ri; Yun, Mijin; Kang, Hyungu; Lee, Jung Hwan; Lim, Jong Hoon; Cho, Won Gil; Kang, Won Jun

    2016-09-01

    Aptamers are promising next-generation ligands used in molecular imaging and theragnosis. Aptamers are synthetic nucleic acids that can be held together with complementary sequences by base-pair hybridization. In this study, the complementary oligonucleotide (cODN) hybridization-based aptamer conjugation platform was developed to use aptamers as the molecular imaging agent. The cODN was pre-labeled with fluorescent dye or radioisotope and hybridized with a matched sequence containing aptamers in aqueous conditions. The cODN platform-hybridized aptamers exhibited good serum stability and specific binding affinity towards target cancer cells both in vitro and in vivo. These results suggest that the newly designed aptamer conjugation platform offers great potential for the versatile application of aptamers as molecular imaging agents. PMID:27258484

  3. Methods To Identify Aptamers against Cell Surface Biomarkers

    OpenAIRE

    Frédéric Ducongé; Daniel Miotto Dupont; Agnes Cibiel

    2011-01-01

    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 cytometr...

  4. Inhibiting heat shock factor 1 in human cancer cells with a potent RNA aptamer.

    Directory of Open Access Journals (Sweden)

    H Hans Salamanca

    Full Text Available Heat shock factor 1 (HSF1 is a master regulator that coordinates chaperone protein expression to enhance cellular survival in the face of heat stress. In cancer cells, HSF1 drives a transcriptional program distinct from heat shock to promote metastasis and cell survival. Its strong association with the malignant phenotype implies that HSF1 antagonists may have general and effective utilities in cancer therapy. For this purpose, we had identified an avid RNA aptamer for HSF1 that is portable among different model organisms. Extending our previous work in yeast and Drosophila, here we report the activity of this aptamer in human cancer cell lines. When delivered into cells using a synthetic gene and strong promoter, this aptamer was able to prevent HSF1 from binding to its DNA regulation elements. At the cellular level, expression of this aptamer induced apoptosis and abolished the colony-forming capability of cancer cells. At the molecular level, it reduced chaperones and attenuated the activation of the MAPK signaling pathway. Collectively, these data demonstrate the advantage of aptamers in drug target validation and support the hypothesis that HSF1 DNA binding activity is a potential target for controlling oncogenic transformation and neoplastic growth.

  5. Rapid capacitive detection of femtomolar levels of bisphenol A using an aptamer-modified disposable microelectrode array

    International Nuclear Information System (INIS)

    A label-free and single-step method is reported for rapid and highly sensitive detection of bisphenol A (BPA) in aqueous samples. It utilizes an aptamer acting as a probe molecule immobilized on a commercially available array of interdigitated aluminum microelectrodes. BPA was quantified by measuring the interfacial capacitance change rate caused by the specific binding between bisphenol A and the immobilized aptamer. The AC signal also induces an AC electrokinetic effect to generate microfluidic motion for enhanced binding. The capacitive aptasensor achieves a limit of detection as low as 10 fM(2.8 fg ⋅ mL−1) with a 20 s response time. The method is inexpensive, highly sensitive, rapid and therefore provides a promising technology for on-site detection of BPA in food and water samples. (author)

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

    Directory of Open Access Journals (Sweden)

    Annamaria eRuscito

    2016-05-01

    Full Text Available 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. 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

  8. 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

  9. 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.

  10. Development of aptamer-based radiopharmaceuticals for targeted cancer imaging and therapy

    OpenAIRE

    Gijs, Marlies; Aerts, An; Impens, Nathalie; Baatout, Sarah; Luxen, André

    2013-01-01

    This SELEX experiment, for the selection of HER2 targeting aptamers, resulted in 26 selected RNA aptamers for further individual evaluation. Up till now, we were able to identify two aptamers that show binding to HER2 overexpressing SK-OV-3 cancer cells. In addition, we were able to generate silenced SK-OV-3 cells with minimal remaining HER2 levels, which will be used to obtain more information regarding the binding specificity of the selected aptamers.

  11. Spatial recognition and mapping of proteins using DNA aptamers

    Science.gov (United States)

    Wang, Congzhou; Yadavalli, Vamsi K.

    2014-11-01

    Atomic force microscopy-based adhesion force measurements have emerged as a powerful tool for the biophysical analyses of biological systems. Such measurements can now be extended to detection and mapping of biomolecules on surfaces via integrated imaging and force spectroscopy techniques. Critical to these experiments is the choice of the biomolecular recognition probe. In this study, we demonstrate how oligonucleotide aptamers can be used as versatile probes to simultaneously image and spatially locate targets on surfaces. We focus on two structurally distinct proteins relevant to the clotting cascade—human α-thrombin and vascular endothelial growth factor. Via AFM-recognition mapping using specific DNA aptamers on a commercially available instrument, we show a clear consistency between height and force measurements obtained simultaneously. Importantly, we are able to observe changes in binding due to changes in the external microenvironment, which demonstrate the ability to study fluctuating biological systems in real time. The aptamer specificity and the ability to distinguish their targets are shown through positive and negative controls. It is therefore possible to generate high resolution maps to spatially and temporally identify proteins at the molecular level on complex surfaces.

  12. Spatial recognition and mapping of proteins using DNA aptamers

    International Nuclear Information System (INIS)

    Atomic force microscopy-based adhesion force measurements have emerged as a powerful tool for the biophysical analyses of biological systems. Such measurements can now be extended to detection and mapping of biomolecules on surfaces via integrated imaging and force spectroscopy techniques. Critical to these experiments is the choice of the biomolecular recognition probe. In this study, we demonstrate how oligonucleotide aptamers can be used as versatile probes to simultaneously image and spatially locate targets on surfaces. We focus on two structurally distinct proteins relevant to the clotting cascade—human α-thrombin and vascular endothelial growth factor. Via AFM-recognition mapping using specific DNA aptamers on a commercially available instrument, we show a clear consistency between height and force measurements obtained simultaneously. Importantly, we are able to observe changes in binding due to changes in the external microenvironment, which demonstrate the ability to study fluctuating biological systems in real time. The aptamer specificity and the ability to distinguish their targets are shown through positive and negative controls. It is therefore possible to generate high resolution maps to spatially and temporally identify proteins at the molecular level on complex surfaces. (paper)

  13. In vitro selection of DNA aptamers binding pesticide fluoroacetamide.

    Science.gov (United States)

    Cao, Fangqi; Lu, Xinwei; Hu, Xiaolong; Zhang, Yurong; Zeng, Libo; Chen, Liankang; Sun, Meiqi

    2016-05-01

    Fluoroacetamide (Mw = 77.06) is a lethal rodenticide to humans and animals which is still frequently abused in food storage somewhere in China. The production of antibodies for fluoroacetamide is difficult due to its high toxicity to animals, which limits the application of immunoassay method in poison detection. In this work, aptamers targeting N-fluoroacetyl glycine as an analog of fluoroacetamide were selected by a specific systematic evolution of ligands by exponential enrichment (SELEX) strategy. The binding ability of the selected aptamers to fluoroacetamide was identified using surface plasmon resonance (SPR)-based assay. The estimated KD values in the low micromolar range showed a good affinity of these aptamers to the target. Our work verified that the SELEX strategy has the potential for developing aptamers targeted to small molecular toxicants and aptamers can be employed as new recognition elements instead of antibodies for poison detection. PMID:26873572

  14. 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.

  15. 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. PMID:26139320

  16. Monitoring Intact Viruses Using Aptamers.

    Science.gov (United States)

    Kumar, Penmetcha K R

    2016-01-01

    Viral diagnosis and surveillance are necessary steps in containing the spread of viral diseases, and they help in the deployment of appropriate therapeutic interventions. In the past, the commonly employed viral detection methods were either cell-culture or molecule-level assays. Most of these assays are laborious and expensive, require special facilities, and provide a slow diagnosis. To circumvent these limitations, biosensor-based approaches are becoming attractive, especially after the successful commercialization of glucose and other biosensors. In the present article, I have reviewed the current progress using the biosensor approach for detecting intact viruses. At the time of writing this review, three types of bioreceptor surfaces (antibody-, glycan-, and aptamer-based) have been explored on different sensing platforms for detecting intact viruses. Among these bioreceptors, aptamer-based sensors have been increasingly explored for detecting intact viruses using surface plasmon resonance (SPR) and other platforms. Special emphasis is placed on the aptamer-based SPR platform in the present review. PMID:27527230

  17. Monitoring Intact Viruses Using Aptamers

    Directory of Open Access Journals (Sweden)

    Penmetcha K. R. Kumar

    2016-08-01

    Full Text Available Viral diagnosis and surveillance are necessary steps in containing the spread of viral diseases, and they help in the deployment of appropriate therapeutic interventions. In the past, the commonly employed viral detection methods were either cell-culture or molecule-level assays. Most of these assays are laborious and expensive, require special facilities, and provide a slow diagnosis. To circumvent these limitations, biosensor-based approaches are becoming attractive, especially after the successful commercialization of glucose and other biosensors. In the present article, I have reviewed the current progress using the biosensor approach for detecting intact viruses. At the time of writing this review, three types of bioreceptor surfaces (antibody-, glycan-, and aptamer-based have been explored on different sensing platforms for detecting intact viruses. Among these bioreceptors, aptamer-based sensors have been increasingly explored for detecting intact viruses using surface plasmon resonance (SPR and other platforms. Special emphasis is placed on the aptamer-based SPR platform in the present review.

  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. 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.

  20. 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.

  1. Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch

    OpenAIRE

    Villa, Alessandra; Wöhnert, Jens; Stock, Gerhard

    2009-01-01

    Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA's secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in...

  2. Optical Nanosensor Architecture for Cell-Signaling Molecules Using DNA Aptamer-Coated Carbon Nanotubes

    OpenAIRE

    Cha, Tae-Gon; Baker, Benjamin A.; Sauffer, M. Dane; Salgado, Janette; Jaroch, David; Rickus, Jenna; Porterfield, D. Marshall; Choi, Jong Hyun

    2011-01-01

    We report a novel optical biosensor platform using near-infrared fluorescent single-walled carbon nanotubes (SWNTs) functionalized with target-recognizing aptamer DNA for noninvasively detecting cell-signaling molecules In real time. Photoluminescence (PL) emission of aptamer-coated SWNTs is modulated upon selectively binding to target molecules, which is exploited to detect insulin using an insulin-binding aptamer (IBA) as a molecular recognition element. We find that nanotube PL quenches up...

  3. Targeting of Antibodies using Aptamers

    OpenAIRE

    Missailidis, Sotiris

    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.

  4. Discrete atomic layers at the molecular level

    International Nuclear Information System (INIS)

    In this review, we deal with the syntheses of large discrete atomic layers at the molecular level. Spectroscopic measurements as well as X-ray crystallographic analyses lead to unambiguous characterizations of these layers. The molecular atomic layers can be considered to be parts of graphenes and related atomic layers, thereby helping to understand such indefinitely huge atomic layers or serving as seeds for the controlled synthesis of nanocarbons. (author)

  5. Study on the specific interaction between angiogenin and aptamer by atomic force microscopy (AFM)

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The specific interaction between angiogenin and aptamer has been investigated by using AFM. The specificity of the interaction is revealed by comparing the binding probability of aptamer to other elements in a series of control experiments.The results have shown that there is specific interaction force between angiogenin and aptamer. Moreover, the single molecular pull-off force between angiogenin and aptamer has also been determined using the Poisson statistical method to be 133.7±11.7 pN. These findings obtained are helpful to the better revelation of recognition mechanism between angiogenin and aptamer, which provided basis for further understanding the inhibition of the aptamer to angiogenic activity.

  6. Current Status and Future Prospects for Aptamer-Based Mycotoxin Detection.

    Science.gov (United States)

    Ruscito, Annamaria; Smith, McKenzie; Goudreau, Daniel N; DeRosa, Maria C

    2016-07-01

    Aptamers are single-stranded oligonucleotides with the ability to bind tightly and selectively to a target analyte. High-affinity and specific aptamers for a variety of mycotoxins have been reported over the past decade. Increasingly, these molecular recognition elements are finding applications in biosensors and assays for the detection of mycotoxins in a variety of complex matrixes. This review article highlights the mycotoxin aptamers that are available for mycotoxin detection and the array of biosensing platforms into which they have been incorporated. Key advantages that aptamers have over analogous technology, and areas in which these advantages may be applied for the benefit of practical mycotoxin detection, are also discussed. PMID:27318356

  7. Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches

    NARCIS (Netherlands)

    Ruigrok, Vincent J. B.; Levisson, Mark; Hekelaar, Johan; Smidt, Hauke; Dijkstra, Bauke W.; van der Oost, John

    2012-01-01

    Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the

  8. Analysis and Identification of Aptamer-Compound Interactions with a Maximum Relevance Minimum Redundancy and Nearest Neighbor Algorithm

    Directory of Open Access Journals (Sweden)

    ShaoPeng Wang

    2016-01-01

    Full Text Available The development of biochemistry and molecular biology has revealed an increasingly important role of compounds in several biological processes. Like the aptamer-protein interaction, aptamer-compound interaction attracts increasing attention. However, it is time-consuming to select proper aptamers against compounds using traditional methods, such as exponential enrichment. Thus, there is an urgent need to design effective computational methods for searching effective aptamers against compounds. This study attempted to extract important features for aptamer-compound interactions using feature selection methods, such as Maximum Relevance Minimum Redundancy, as well as incremental feature selection. Each aptamer-compound pair was represented by properties derived from the aptamer and compound, including frequencies of single nucleotides and dinucleotides for the aptamer, as well as the constitutional, electrostatic, quantum-chemical, and space conformational descriptors of the compounds. As a result, some important features were obtained. To confirm the importance of the obtained features, we further discussed the associations between them and aptamer-compound interactions. Simultaneously, an optimal prediction model based on the nearest neighbor algorithm was built to identify aptamer-compound interactions, which has the potential to be a useful tool for the identification of novel aptamer-compound interactions. The program is available upon the request.

  9. Active Site Analysis and Modification of Organophosphorus Pesticides Aptamers Based on Molecular Beacon%基于分子信标的有机磷农药适配体活性位点分析及改造

    Institute of Scientific and Technical Information of China (English)

    王丽; 张存政; 刘媛; 屠康; 桑宏庆; 刘贤进

    2012-01-01

    A competitive inhibition method based on a molecular beacon containing 20 nucleotides was developed for the active site analysis and modification of two organophosphorusate pesticides aptamers that had been selected in our previous study. The results indicated that the designed molecular beacon formed a hairpin structure, and the loop could be opened up in the presence of complementary aptamer sequence. The ratio of molecular beacon to aptamer of 1. 25 to 1, incubated time of 50 min and incubation at room temperature were chosen as optimal conditions of active site analysis. Under optimal conditions, the Loop2-4 was a mutual active site for four organophosphorus pesticides, Loop2-3 and the nucleotides at 3' and 5' of the SS4-54 aptamer were important active sites for phorate, Loop2-2 and Loop4-2 were mutual active sites for profenofos and isocarbophos, Loop4-3 was a mutual active site for profenofos and omethoate, Loop2-1 and Loop4-1 were important active sites for isocarbophos. The binding activity for profenofos and isocarbophos of SS24-PJ-35 aptamers modified by gene splicing significantly increased.%设计了一个长度为20个核苷酸的分子信标,建立了有机磷农药和分子信标竞争结合适配体鉴定其活性的方法,对前期筛选的两条适配体进行了活性位点分析和改造.结果表明,分子信标设计合理,性能稳定,其发夹结构在室温下既可成功闭合也可成功打开,最佳的活性鉴定条件为分子信标与适配体添加比例1.25∶1,孵育时间50 min,孵育温度为室温.活性位点分析表明Loop2-4是4种有机磷农药共有的活性位点,Loop2-3及SS4-54适配体5’端和3 '端残余的核苷酸是甲拌磷重要的活性位点,Loop2-2和Loop4-2是丙溴磷和水胺硫磷共有的活性位点,Loop4-3是丙溴磷和氧化乐果共有的活性位点,Loop2-1和Loop4-1是水胺硫磷重要的活性位点.通过基因拼接改造的SS24-PJ-35适配体对丙溴磷和水胺硫磷的结合活性明显提高.

  10. 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.

  11. Development of a novel DNA aptamer ligand targeting to primary cultured tumor endothelial cells by a cell-based SELEX method.

    Directory of Open Access Journals (Sweden)

    Mst Naznin Ara

    Full Text Available The present study used a spontaneous cell-based SELEX method (Systemic Evolution of Ligands by EXponential Enrichment to produce DNA aptamers that specifically bind to cell surface proteins or biomarkers produced by primary cultured mouse tumor endothelial cells (mTECs. In solid tumors, new blood vessels are formed through an angiogenesis process, and this plays a critical role in cancer development as well as metastasis. To combat angiogenesis, an appropriate diagnosis and a molecular-level understanding of the different cancer types are now a high priority. The novel DNA aptamer AraHH001, developed in this study, binds specifically to mTECs with high affinity in the nano-molar range, but does not bind to normal skin endothelial cells (skin-ECs. The selected DNA aptamer was also found to bind to cultured human tumor endothelial cells (hTECs, isolated from a clinical patient with a renal carcinoma. The aptamer AraHH001 showed significant anti-angiogenesis activity by inhibiting tube formation by mTECs on matrigel. Interestingly, a confocal laser scanning microscopy examination of in vitro cellular uptake revealed that AraHH001 was assimilated by mTECs, and became co-localized in acidic compartments, as detected by labeling with Lysotracker Red. Therefore, the development of a specific DNA aptamer that binds to mTECs, as reported here for the first time, holds great promise not only as a therapeutic aptamer but also as a targeted molecular probe that appears to play a major role in angiogenesis, and for the development of a targeted new drug delivery system.

  12. 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.

  13. Aptamers and Their Biological Applications

    Directory of Open Access Journals (Sweden)

    Changill Ban

    2012-01-01

    Full Text Available Recently, aptamers have attracted the attention of many scientists, because they not only have all of the advantages of antibodies, but also have unique merits, such as thermal stability, low cost, and unlimited applications. In this review, we present the reasons why aptamers are known as alternatives to antibodies. Furthermore, several types of in vitro selection processes, including nitrocellulose membrane filtration, affinity chromatography, magnetic bead, and capillary electrophoresis-based selection methods, are explained in detail. We also introduce various applications of aptamers for the diagnosis of diseases and detection of small molecules. Numerous analytical techniques, such as electrochemical, colorimetric, optical, and mass-sensitive methods, can be utilized to detect targets, due to convenient modifications and the stability of aptamers. Finally, several medical and analytical applications of aptamers are presented. In summary, aptamers are promising materials for diverse areas, not just as alternatives to antibodies, but as the core components of medical and analytical equipment.

  14. 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.

  15. Aptamers: Problems, Solutions and Prospects

    OpenAIRE

    Lakhin, A.; Tarantul, V.; Gening, L.

    2013-01-01

    Aptamers are short single-stranded oligonucleotides that are capable of binding various molecules with high affinity and specificity. When the technology of aptamer selection was developed almost a quarter of a century ago, a suggestion was immediately put forward that it might be a revolutionary start into solving many problems associated with diagnostics and the therapy of diseases. However, multiple attempts to use aptamers in practice, although sometimes successful, have been generally mu...

  16. 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...

  17. High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

    International Nuclear Information System (INIS)

    We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be KD = 2.25x10-9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

  18. In vivo imaging of oligonucleotidic aptamers

    Energy Technology Data Exchange (ETDEWEB)

    Tavitian, B.; Boisgard, R. [Inserm U803, Laboratoire d' Imagerie Moleculaire Experimentale, Service hospitalier Frederic joliot, Intitut d' Imagerie Biomedicale, CEA, Orsay (France); Duconge, F.; Dolle, F. [Groupe de Radiochimie, Laboratoire d' Imagerie Moleculaire Experimentale, Service hospitalier Frederic joliot, Intitut d' Imagerie Biomedicale, CEA, Orsay (France)

    2009-07-01

    In this chapter we present the methods developed in our laboratory for in vivo imaging of oligonucleotidic aptamers. These methods relate to (i) the labelling of aptamers with fluorine-18, a positron emitter (ii) Positron Emission Tomography imaging of laboratory animals with [({sup 18})F]aptamers and (iii) labelling with fluorescent dyes and optical imaging of aptamers in mice. (authors)

  19. 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.

  20. Methods for Improving Aptamer Binding Affinity

    OpenAIRE

    Hijiri Hasegawa; Nasa Savory; Koichi Abe; Kazunori Ikebukuro

    2016-01-01

    Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of a...

  1. Design of Potent and Controllable Anticoagulants Using DNA Aptamers and Nanostructures

    Directory of Open Access Journals (Sweden)

    Abhijit Rangnekar

    2016-02-01

    Full Text Available The regulation of thrombin activity offers an opportunity to regulate blood clotting because of the central role played by this molecule in the coagulation cascade. Thrombin-binding DNA aptamers have been used to inhibit thrombin activity. In the past, to address the low efficacy reported for these aptamers during clinical trials, multiple aptamers have been linked using DNA nanostructures. Here, we modify that strategy by linking multiple copies of various thrombin-binding aptamers using DNA weave tiles. The resulting constructs have very high anticoagulant activity in functional assays owing to their improved cooperative binding affinity to thrombin due to optimized spacing, orientation, and the high local concentration of aptamers. We also report the results of molecular dynamics simulations to gain insight into the solution conformations of the tiles. Moreover, by using DNA strand displacement, we were able to turn the coagulation cascade off and on as desired, thereby enabling significantly better control over blood coagulation.

  2. Binding modes of thrombin binding aptamers investigated by simulations and experiments

    Science.gov (United States)

    Trapaidze, A.; Bancaud, A.; Brut, M.

    2015-01-01

    Thrombin binding aptamers HD1 and HD22 are the most studied aptamers, both for therapeutic and sensing purposes. Yet, there is still no commercialized aptamer-based sensor device for thrombin detection, suggesting that the binding modes of these aptamers remain to be precisely described. Here, we investigate thrombin-aptamer interactions with molecular dynamics simulations, and show that the different solved structures of HD1-thrombin complex are energetically similar and consequently possibly co-existing. Conversely, HD22 folding is much more stable, and its binding energy with thrombin is significantly larger than that of HD1 complexes. These results are confronted to experiments, which consist in monitoring aggregation of aptamer-functionalized gold nanoparticles triggered by thrombin. HD1 alone, but not HD22, can trigger aggregation, meaning that this aptamer has multiple sites of interactions with thrombin. Furthermore, pre-incubation of HD22 with thrombin impedes HD1 aggregation, suggesting that HD1 and HD22 have competing affinities for the same binding site. Altogether, this study shows that the characterization of aptamer-thrombin interactions by structural and kinetic experiments joined to simulations is necessary for the development of biosensors.

  3. A DNA nanocapsule with aptamer-controlled open-closure function for targeted delivery

    DEFF Research Database (Denmark)

    Bentin, Thomas

    2012-01-01

    A DNA capsule fitted with aptamer controlled target sensing has been "woven" using a 7308-base single-stranded DNA "thread" and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces.......A DNA capsule fitted with aptamer controlled target sensing has been "woven" using a 7308-base single-stranded DNA "thread" and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces....

  4. 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

    2016-01-01

    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. PMID:27438862

  5. Leukemia Diagnosis and Aptamer Technology%白血病诊断与适配体技术

    Institute of Scientific and Technical Information of China (English)

    孙娜; 夏薇

    2013-01-01

    Aptamer, a high specificity of oligonucleotide ligand gained through systematic evolution of ligands by exponential enrichment ( SELEX) , is a new type of molecular probes. The application of this technology has successfully screened acute myeloid leukemia and other leukemia cell-specific aptamer in vitro. It also provides the possibility to establish a simple,rapid,accurate diagnosis of leukemia at the molecular level.%通过指数富集的配体系统进化技术(SELEX)能够获得特异性的寡核苷酸配体,即核酸适配体(Aptamer).Aptamer是一类新型的分子探针,应用该技术已经成功在体外筛选出急性髓系白血病等白血病细胞特异性适配体,为从分子水平建立一种简便、快速、精准的白血病诊断新方法提供了可能.

  6. Evolution of DNA aptamers for malignant brain tumor gliosarcoma cell recognition and clinical tissue imaging.

    Science.gov (United States)

    Wu, Qiaoyi; Wu, Liang; Wang, Yuzhe; Zhu, Zhi; Song, Yanling; Tan, Yuyu; Wang, Xing-Fu; Li, Jiuxing; Kang, Dezhi; Yang, Chaoyong James

    2016-06-15

    Gliosarcoma, a variant of glioblastoma multiforme (GBM), is a highly invasive malignant tumor. Unfortunately, this disease still marked by poor prognosis regardless of modern treatments. It is of great significance to discover specific molecular probes targeting gliosarcoma for early cancer diagnosis and therapy. Herein, we have selected a group of DNA aptamers with high affinity and selectivity against gliosarcoma cells K308 using cell-SELEX. All the dissociation constants of these aptamers against gliosarcoma cells were in the nanomolar range and aptamer WQY-9 has the highest affinity and good selectivity among them. Furthermore, truncated aptamer sequence, WQY-9-B, shows similar recognition ability to aptamer WQY-9. In addition, WQY-9-B was found to be able to bind selectively and internalize into cytoplasm of target cancer cell at 37°C. More importantly, compared to a random sequence, aptamer WQY-9-B showed excellent recognition rate (73.3%) for tissue sections of clinical gliosarcoma samples. These data suggests that aptamer WQY-9-B has excellent potential as an effective molecular probe for gliosarcoma diagnosis. PMID:26802746

  7. 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.

  8. Aptamers and Their Biological Applications

    OpenAIRE

    Changill Ban; Seonghwan Lee; Kyung-Mi Song

    2012-01-01

    Recently, aptamers have attracted the attention of many scientists, because they not only have all of the advantages of antibodies, but also have unique merits, such as thermal stability, low cost, and unlimited applications. In this review, we present the reasons why aptamers are known as alternatives to antibodies. Furthermore, several types of in vitro selection processes, including nitrocellulose membrane filtration, affinity chromatography, magnetic bead, and capillary electrophoresis-ba...

  9. Targeting cancer with peptide aptamers

    OpenAIRE

    Seigneuric, Renaud; Gobbo, Jessica; Colas, Pierre; Garrido, Carmen

    2011-01-01

    A major endeavour in cancer chemotherapy is to develop agents that specifically target a biomolecule of interest. There are two main classes of targeting agents: small molecules and biologics. Among biologics (e.g.: antibodies), DNA, RNA but also peptide aptamers are relatively recent agents. Peptide aptamers are seldom described but represent attractive agents that can inhibit a growing panel of oncotargets including Heat Shock Proteins. Potential pitfalls and coming challenges towards succe...

  10. Nucleic Acid Aptamers Against Proteases

    OpenAIRE

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

    2011-01-01

    Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of...

  11. Aptamers for Targeted Drug Delivery

    Directory of Open Access Journals (Sweden)

    Partha Ray

    2010-05-01

    Full Text Available Aptamers are a class of therapeutic oligonucleotides that form specific three-dimensional structures that are dictated by their sequences. They are typically generated by an iterative screening process of complex nucleic acid libraries employing a process termed Systemic Evolution of Ligands by Exponential Enrichment (SELEX. SELEX has traditionally been performed using purified proteins, and cell surface receptors may be challenging to purify in their properly folded and modified conformations. Therefore, relatively few aptamers have been generated that bind cell surface receptors. However, improvements in recombinant fusion protein technology have increased the availability of receptor extracellular domains as purified protein targets, and the development of cell-based selection techniques has allowed selection against surface proteins in their native configuration on the cell surface. With cell-based selection, a specific protein target is not always chosen, but selection is performed against a target cell type with the goal of letting the aptamer choose the target. Several studies have demonstrated that aptamers that bind cell surface receptors may have functions other than just blocking receptor-ligand interactions. All cell surface proteins cycle intracellularly to some extent, and many surface receptors are actively internalized in response to ligand binding. Therefore, aptamers that bind cell surface receptors have been exploited for the delivery of a variety of cargoes into cells. This review focuses on recent progress and current challenges in the field of aptamer-mediated delivery.

  12. Selection of a high-affinity and in vivo bioactive ssDNA aptamer against angiotensin II peptide.

    Science.gov (United States)

    Heiat, Mohammad; Ranjbar, Reza; Latifi, Ali Mohammad; Rasaee, Mohammad Javad

    2016-08-01

    Unique features of aptamers have attracted interests for a broad range of applications. Aptamers are able to specifically bind to targets and inhibit their functions. This study, aimed to isolate the high affinity ssDNA aptamers against bio-regulator peptide angiotensin II (Ang II) and investigate their bioactivity in cellular and animal models. To isolate ssDNA aptamers, 12 rounds of affinity chromatography SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure were carried out. The SPR (surface plasmon resonance) and ELONA (enzyme linked oligonucleotide assay) analysis were used to determine the affinity and specificity of aptamers. The ability of selected aptamers to inhibit the proliferative effect of Ang II on human aortic vascular smooth muscle cells (HA-VSMCs) and their performance on Wistar rat urinary system and serum electrolyte levels were investigated. Two full-length aptamers (FLC112 and FLC125) with high affinity of respectively 7.52±2.44E-10 and 5.87±1.3E-9M were isolated against Ang II. The core regions of these aptamers (CRC112 and CRC125) also showed affinity of 5.33±1.15E-9 and 4.11±1.09E-9M. In vitro analysis revealed that FLC112 and FLC125 can inhibit the proliferative effect of Ang II on HA-VSMCs (Psodium level and increased the urine volume (Pbioactive aptamers may lead to excellent results in blocking Ang II activity. PMID:27298205

  13. Aptamers in Virology: Recent Advances and Challenges

    OpenAIRE

    Jennifer M. Binning; 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...

  14. Cell-Specific Aptamers as Emerging Therapeutics

    OpenAIRE

    Cindy Meyer; Ulrich Hahn; Andrea Rentmeister

    2011-01-01

    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...

  15. Inhibition of hepatitis C virus infection by DNA aptamer against NS2 protein.

    Science.gov (United States)

    Gao, Yimin; Yu, Xiaoyan; Xue, Binbin; Zhou, Fei; Wang, Xiaohong; Yang, Darong; Liu, Nianli; Xu, Li; Fang, Xiaohong; Zhu, Haizhen

    2014-01-01

    NS2 protein is essential for hepatitis C virus (HCV) replication. NS2 protein was expressed and purified. Aptamers against NS2 protein were raised and antiviral effects of the aptamers were examined. The molecular mechanism through which the aptamers exert their anti-HCV activity was investigated. The data showed that aptamer NS2-3 inhibited HCV RNA replication in replicon cell line and infectious HCV cell culture system. NS2-3 and another aptamer NS2-2 were demonstrated to inhibit infectious virus production without cytotoxicity in vitro. They did not affect hepatitis B virus replication. Interferon beta (IFN-β) and interferon-stimulated genes (ISGs) were not induced by the aptamers in HCV-infected hepatocytes. Furthermore, our study showed that N-terminal region of NS2 protein is involved in the inhibition of HCV infection by NS2-2. I861T within NS2 is the major resistance mutation identified. Aptamer NS2-2 disrupts the interaction of NS2 with NS5A protein. The data suggest that NS2-2 aptamer against NS2 protein exerts its antiviral effects through binding to the N-terminal of NS2 and disrupting the interaction of NS2 with NS5A protein. NS2-specific aptamer is the first NS2 inhibitor and can be used to understand the mechanisms of virus replication and assembly. It may be served as attractive candidates for inclusion in the future HCV direct-acting antiviral combination therapies. PMID:24587329

  16. Identification of Staphylococcus aureus infection by aptamers directly radiolabeled with technetium-99m

    International Nuclear Information System (INIS)

    Introduction: Aptamers are oligonucleotides that have high affinity and specificity for their molecular targets which are emerging as a new class of molecules for radiopharmaceuticals development. In this study, aptamers selected to Staphylococcus aureus were evaluated for bacterial infection identification. Methods: Anti S. aureus aptamers were labeled with 99mTc by the direct method. The radiolabel yield and complex stability were assessed by thin-layer chromatography (TLC). Three groups of Swiss mice containing 6 animals each were used. The first group was infected intramuscularly in the right thigh with S. aureus. The second group was infected in the same way with C. albicans and the third group was injected with zymosan to induce aseptic inflammation. After 24 h, radiolabeled aptamers (22.2 MBq) were injected by the tail vein. The mice were euthanized 4 h post injection and tissue sample activities measured in a gamma counter. Results: The 99mTc labeled aptamers were stable in saline, plasma and cystein excess. Radiolabeled aptamers showed increased uptake in the kidneys for all groups indicating a main renal excretion, which is consistent with the hydrophilic nature and small size of aptamers. The radiopharmaceutical showed rapid blood clearance indicated by a reduced dose (% ID/g) in the blood. The biodistribution showed that aptamers were able to identify the infection foci caused by S. aureus displaying a target/non-target ratio of 4.0 ± 0.5. This ratio for mice infected with C. albicans was 2.0 ± 0.4 while for mice with aseptic inflammation was 1.2 ± 0.2. Histology confirmed the presence of infection in groups 1 and 2, and inflammation in group 3. Conclusions: The biodistibution study demonstrated a statistically higher uptake in the S. aureus foci relative to inflammation and C. albicans infected areas. These results highlight the potential of aptamers labeled directly with 99mTc for bacterial infection diagnosis by scintigraphy

  17. Radiation damage at the molecular level: Nanodosimetry

    International Nuclear Information System (INIS)

    One of the main practical use of the model is its use as a tool of nanodosimetry which basically consists in characterizing the effect of radiation on nano volumes (comparable to the DNA of volumes) in terms of link breaks and molecular dissociations. (Author)

  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. Selection and characterization of a novel DNA aptamer for label-free fluorescence biosensing of ochratoxin A.

    Science.gov (United States)

    McKeague, Maureen; Velu, Ranganathan; Hill, Kayla; Bardóczy, Viola; Mészáros, Tamás; DeRosa, Maria C

    2014-08-01

    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. PMID:25153252

  20. 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.

  1. 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.

  2. 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.

  3. Mirror symmetry breaking at the molecular level.

    OpenAIRE

    Avetisov, V; Goldanskii, V.

    1996-01-01

    Reasoning from two basic principles of molecular physics, P invariance of electromagnetic interaction and the second law of thermodynamics, one would conclude that mirror symmetry retained in the world of chiral molecules. This inference is fully consistent with what is observed in inorganic nature. However, in the bioorganic world, the reverse is true. Mirror symmetry there is definitely broken. Is it possible to account for this phenomenon without going beyond conventional concepts of the k...

  4. Aptamer-conjugated dendrimer-modified quantum dots for glioblastoma cells imaging

    Energy Technology Data Exchange (ETDEWEB)

    Li Zhiming; Huang Peng; He Rong; Bao Chenchen; Cui Daxiang [National Key Laboratory of Nano/Micro Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Institute of Micro-Nano Science and Technology, Shanghai Jiao Tong University, Shanghai 200240 (China); Zhang Xiaomin; Ren Qiushi [Institute for Laser Medicine and Biophotonics, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240 (China)], E-mail: qsren@sjtu.edu.cn, E-mail: dxcui@sjtu.edu.cn

    2009-09-01

    Targeted quantum dots have shown potential as a platform for development of cancer imaging. Aptamers have recently been demonstrated as ideal candidates for molecular targeting applications. In present work, polyamidoamine dendrimers were used to modify surface of quantum dots and improve their solubility in water solution. Then, dendrimer-modified quantum dots were conjugated with DNA aptamer, GBI-10, can recognize the extracellular matrix protein tenascin-C on the surface of human glioblastoma cells. The dendrimer-modified quantum dots exhibit water-soluble, high quantum yield, and good biocompatibility. Aptamer-conjugated quantum dots can specifically target U251 human glioblastoma cells. High-performance aptamer-conjugated dendrimers modified quantum dot-based nanoprobes have great potential in application such as cancer imaging.

  5. Selection of Natural and Base-Modified DNA Aptamers for a Camptothecin Derivative.

    Science.gov (United States)

    Fujita, Hiroto; Kuwahara, Masayasu

    2016-01-01

    Nucleic acid aptamers for small molecules are currently being developed and have a potential role in diverse applications including biosensing, diagnostics, and therapeutics involving low-molecular-weight biomarkers and drugs. To enhance and broaden their functions through chemical modification, systematic evolution of ligands by exponential enrichment (SELEX) selection has been attempted with modified DNA/RNA libraries. Recently, we demonstrated the superior efficacy of base modification for affinity enhancement and the usefulness of unnatural nucleic acid libraries for development of small-molecule aptamers. In this unit, we describe construction of a modified DNA library that includes (E)-5-(2-(N-(2-(N(6) -adeninyl)ethyl))carbamylvinyl)uracil bases and acquisition of high-affinity camptothecin-binding DNA aptamers, in addition to those of the corresponding natural DNA library and aptamers, using the SELEX method. © 2016 by John Wiley & Sons, Inc. PMID:27248786

  6. An RNA aptamer provides a novel approach for the induction of apoptosis by targeting the HPV16 E7 oncoprotein.

    Directory of Open Access Journals (Sweden)

    Clare Nicol

    Full Text Available BACKGROUND: Human papillomavirus 16 (HPV16 is a high-risk DNA tumour virus, which is a major causative agent of cervical cancer. Cellular transformation is associated with deregulated expression of the E6 and E7 oncogenes. E7 has been shown to bind a number of cellular proteins, including the cell cycle control protein pRb. In this study, RNA aptamers (small, single-stranded oligonucleotides selected for high-affinity binding to HPV16 E7 were employed as molecular tools to further investigate these protein-protein interactions. METHODOLOGY/PRINCIPAL FINDINGS: This study is focused on one aptamer (termed A2. Transfection of this molecule into HPV16-transformed cells resulted in inhibition of cell proliferation (shown using real-time cell electronic sensing and MTT assays due to the induction of apoptosis (as demonstrated by Annexin V/propidium iodide staining. GST-pull down and bead binding assays were used to demonstrate that the binding of A2 required N-terminal residues of E7 known to be involved in interaction with the cell cycle control protein, pRb. Using a similar approach, A2 was shown to disrupt the interaction between E7 and pRb in vitro. Furthermore, transfection of HPV16-transformed cells with A2 appeared to result in the loss of E7 and rise in pRb levels, as observed by immunoblotting. CONCLUSIONS/SIGNIFICANCE: This paper includes the first characterisation of the effects of an E7 RNA aptamer in a cell line derived from a cervical carcinoma. Transfection of cells with A2 was correlated with the loss of E7 and the induction of apoptosis. Aptamers specific for a number of cellular and viral proteins have been documented previously; one aptamer (Macugen is approved for clinical use and several others are in clinical trials. In addition to its role as a molecular tool, A2 could have further applications in the future.

  7. Understanding diseases at a molecular level

    Energy Technology Data Exchange (ETDEWEB)

    Rosev, Tatjana K [Los Alamos National Laboratory

    2008-01-01

    A group of scientists at Los Alamos National Laboratory in 2008 successfully pioneered a microscope able to track protein-sized, hard to see particles in three dimensions. The 3D Tracking Microscope, designed and developed by James H. Werner, Guillaume A. Lessard, Nathan Wells and Peter M. Goodwin of LANL's Center for Integrated Nanotechnologies, won a 2008 R&D 100 award. The team's invention is a unique confocal 3D tracking microscope capable of following the motion of nanometer-sized objects, such as individual molecules, quantum dots, organic fluorophores and single green fluorescent proteins as they zoom through three-dimensional space at rates faster than many intracellular transport processes. The 3D tracking microscope can follow the transport of nanometer-sized particles at micrometer per second rates. This enables researchers to follow individual protein, ribonucleic acid (RNA), or deoxyribonucleic acid (DNA) motion throughout the full three-dimensional volume of a cell to discover the path a particular biomolecule takes, the method it employs to get there and the specific proteins it may be interacting with along the way. In addition to applications in molecular spectroscopy and materials research, the 3D tracking microscope is a powerful tool primarily in the fields of cellular biology and biomedical research, Werner said. 'The 3D tracking microscope will advance our understanding of the molecular basis and kinetics of many diseases, such as cancer, diabetes, or muscular dystrophy,' he said. 'We anticipate the microscope will become a valuable weapon in the arsenal of biomedical researchers who are fighting to find cures for cancer, heart disease and other protein or DNA-based diseases.'

  8. Organophosphorus pesticides detection using broad-specific single-stranded DNA based fluorescence polarization aptamer assay.

    Science.gov (United States)

    Zhang, Cunzheng; Wang, Li; Tu, Zhui; Sun, Xing; He, Qinghua; Lei, Zhaojing; Xu, Chongxin; Liu, Yuan; Zhang, Xiao; Yang, Jingyi; Liu, Xianjin; Xu, Yang

    2014-05-15

    An approach is developed to detect the organophosphorus pesticides via competitive binding to a recombinant broad-specificity DNA aptamer with a molecular beacon (MB), the binding of the MB to the aptamer results in the activation of a fluorescent signal, which can be measured for pesticide quantification. Aptamers selected via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) were structurally modified and truncated to narrow down the binding region of the target, which indicated that loops of the aptamer contributed different functions for different chemical recognition. Thereafter, a variant fused by two different minimum functional structures, was clarified with broad specificity and increased affinity. Further molecular docking and molecular dynamics simulations was conducted to understand the molecular interaction between DNA structure and chemicals. 3D modeling revealed a hot spot area formed by 3 binding sites, forces including hydrogen bonds and van der Waals interactions appear to play a significant role in enabling and stabilizing the binding of chemicals. Finally, an engineered aptamer based approach for the detection of organophosphorus pesticides was successfully applied in a test using a real sample, the limit of quantification (LOQ) for phorate, profenofos, isocarbophos, and omethoate reached 19.2, 13.4, 17.2, and 23.4 nM (0.005 mg L(-1)), respectively. PMID:24384262

  9. Aptamer-based radioimmunotherapy. The feasibility and prospect in cancer therapy

    International Nuclear Information System (INIS)

    Radioimmunotherapy (RIT) has emerged as an attractive and promising strategy for the management of malignant diseases. It has been proven to be quite effective in the treatment of numerous tumors, such as non-Hodgkin lymphoma, metastatic prostate cancer, melanoma, thyroid cancer, colon cancer and so on. The RIT currently used is mainly based on monoclonal antibodies to recognize target antigens. As antibodies are large molecules, this method of RIT has some limitations in in vivo use, such as the immunogenicity, the high costs and low efficiency of production. Aptamer is discovered and selected by SELEX technology. As specific recognizers and binders, aptamers and antibodies have such a close similarity as to be interchangeable to some extent. But, aptamers have many advantages over antibodies: higher affinity and specificity, smaller molecular weight, more easily synthesized and modified, more rapidly penetrating into tumors, higher tumor-to-blood distribution ratio and more easily to be cleared. In addition, since aptamer has almost no immunogenicity in vivo, it can be repeatedly administered. Thus, we believe that aptamer-based RIT will be a feasible and promising way to treat human cancers, and it might display better results in cancer treatment than antibody-based RIT. In conclusion, aptamer-based RIT is hopeful to become a key therapeutics in cancer radiotherapy in the near future. (author)

  10. Engineering Signaling Aptamers That Rely on Kinetic Rather Than Equilibrium Competition.

    Science.gov (United States)

    Du, Yan; Zhen, Shu Jun; Li, Bingling; Byrom, Michelle; Jiang, Yu Sherry; Ellington, Andrew D

    2016-02-16

    During the past decade, aptasensors have largely been designed on the basis of the notion that ligand-modulated equilibration between aptamer conformations could be exploited for sensing. One implementation of this strategy has been to denature the aptamer with an antisense oligonucleotide, wait for dissociation of the antisense oligonucleotide, and stabilize the folded, signaling conformer with a ligand. However, there is a large kinetic barrier associated with releasing the oligonucleotide from the aptamer to again obtain an active, binding conformation. If the length of the antisense oligonucleotide is decreased to make dissociation from the aptamer more favorable, higher background signals are observed. To improve the general methodology for developing aptasensors, we have developed a novel and robust strategy for aptasensor design in which an oligonucleotide kinetically competes with the ligand for binding rather than having to be released from a stable duplex. While the oligonucleotide can induce conformational change, it initially chooses between the aptamer and a molecular beacon (MB), a process that does not require a lengthy pre-equilibration. Using an anti-ricin aptamer as a starting point, we developed a "competitive" aptasensor with a measured limit of detection (LOD) of 30 nM with an optical readout and as low as 3 nM for ricin toxin A-chain (RTA) detection on an electrochemical platform. PMID:26750592

  11. Searching the Sequence Space for Potent Aptamers Using SELEX in Silico.

    Science.gov (United States)

    Zhou, Qingtong; Xia, Xiaole; Luo, Zhaofeng; Liang, Haojun; Shakhnovich, Eugene

    2015-12-01

    To isolate functional nucleic acids that bind to defined targets with high affinity and specificity, which are known as aptamers, the systematic evolution of ligands by exponential enrichment (SELEX) methodology has emerged as the preferred approach. Here, we propose a computational approach, SELEX in silico, that allows the sequence space to be more thoroughly explored regarding binding of a certain target. Our approach consists of two steps: (i) secondary structure-based sequence screening, which aims to collect the sequences that can form a desired RNA motif as an enhanced initial library, followed by (ii) sequence enrichment regarding target binding by molecular dynamics simulation-based virtual screening. Our SELEX in silico method provided a practical computational solution to three key problems in aptamer sequence searching: design of nucleic acid libraries, knowledge of sequence enrichment, and identification of potent aptamers. Six potent theophylline-binding aptamers, which were isolated by SELEX in silico from a sequence space containing 4(13) sequences, were experimentally verified to bind theophylline with high affinity: Kd ranging from 0.16 to 0.52 μM, compared with the dissociation constant of the original aptamer-theophylline, 0.32 μM. These results demonstrate the significant potential of SELEX in silico as a new method for aptamer discovery and optimization. PMID:26642994

  12. 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. PMID:26547431

  13. Aptamer/quantum dot-based simultaneous electrochemical detection of multiple small molecules

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Haixia [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Jiang Bingying [School of Chemistry and Chemical Engineering, Chongqing University of Technology, Chongqing 400040 (China); Xiang Yun, E-mail: yunatswu@swu.edu.cn [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Zhang Yuyong; Chai Yaqin [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Yuan Ruo, E-mail: yuanruo@swu.edu.cn [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China)

    2011-03-04

    A novel strategy for 'signal on' and sensitive one-spot simultaneous detection of multiple small molecular analytes based on electrochemically encoded barcode quantum dot (QD) tags is described. The target analytes, adenosine triphosphate (ATP) and cocaine, respectively, are sandwiched between the corresponding set of surface-immobilized primary binding aptamers and the secondary binding aptamer/QD bioconjugates. The captured QDs yield distinct electrochemical signatures after acid dissolution, whose position and size reflect the identity and level, respectively, of the corresponding target analytes. Due to the inherent amplification feature of the QD labels and the 'signal on' detection scheme, as well as the sensitive monitoring of the metal ions released upon acid dissolution of the QD labels, low detection limits of 30 nM and 50 nM were obtained for ATP and cocaine, respectively, in our assays. Our multi-analyte sensing system also shows high specificity to target analytes and promising applicability to complex sample matrix, which makes the proposed assay protocol an attractive route for screening of small molecules in clinical diagnosis.

  14. Aptamers as Therapeutics in Cardiovascular Diseases

    OpenAIRE

    Wang, Pu; Yang, Yunan; Hong, Hao; Zhang, Yin; Cai, Weibo; Fang, Dianchun

    2011-01-01

    With many advantages over other therapeutic agents such as monoclonal antibodies, aptamers have recently emerged as a novel and powerful class of ligands with excellent potential for diagnostic and therapeutic applications. Typically generated through Systematic Evolution of Ligands by EXponential enrichment (SELEX), aptamers have been selected against a wide range of targets such as proteins, phospholipids, sugars, nucleic acids, as well as whole cells. DNA/RNA aptamers are single-stranded D...

  15. Using Aptamers for Cancer Biomarker Discovery

    OpenAIRE

    Yun Min Chang; Donovan, Michael J; Weihong Tan

    2013-01-01

    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...

  16. Trends in aptamer selection methods and applications

    OpenAIRE

    Yüce, Meral; Yuce, Meral; Ullah, Naimat; Budak, Hikmet

    2015-01-01

    Aptamers are target specific ssDNA, RNA or peptide sequences generated by an in vitro selection and amplification method called SELEX (Systematic Evolution of Ligands by EXponential Enrichment), which involves repetitive cycles of binding, recovery and amplification steps. Aptamers have the ability to bind with a variety of targets such as drugs, proteins, heavy metals, and pathogens with high specificity and selectivity. Aptamers are similar to monoclonal antibodies regarding their binding a...

  17. Aptamers: A Feasible Technology in Cancer Immunotherapy

    OpenAIRE

    Soldevilla, M. M.; H. Villanueva; Pastor, F. (Fernando)

    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 immun...

  18. Therapeutic aptamers: developmental potential as anticancer drugs

    OpenAIRE

    Lee, Ji Won; Kim, Hyun Jung; Heo, Kyun

    2015-01-01

    Aptamers, composed of single-stranded DNA or RNA oligonucleotides that interact with target molecules through a specific three-dimensional structure, are selected from pools of combinatorial oligonucleotide libraries. With their high specificity and affinity for target proteins, ease of synthesis and modification, and low immunogenicity and toxicity, aptamers are considered to be attractive molecules for development as anticancer therapeutics. Two aptamers - one targeting nucleolin and a seco...

  19. 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

  20. Inhibition of BACE1 Activity by a DNA Aptamer in an Alzheimer's Disease Cell Model.

    Directory of Open Access Journals (Sweden)

    Huiyu Liang

    Full Text Available An initial step in amyloid-β (Aβ production includes amyloid precursor protein (APP cleavage via β-Site amyloid precursor protein cleaving enzyme 1 (BACE1. Increased levels of brain Aβ have been implicated in the pathogenesis of Alzheimer's disease (AD. Thus, β-secretase represents a primary target for inhibitor drug development in AD. In this study, aptamers were obtained from combinatorial oligonucleotide libraries using a technology referred to as systematic evolution of ligands by exponential enrichment (SELEX. A purified human BACE1 extracellular domain was used as a target to conduct an in vitro selection process using SELEX. Two DNA aptamers were capable of binding to BACE1 with high affinity and good specificity, with Kd values in the nanomolar range. We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model. We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media. Furthermore, Western blot analysis indicated that sAPPβ expression significantly decreased in the A1 treated versus control groups. These findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.

  1. Post-SELEX optimization of aptamers.

    Science.gov (United States)

    Gao, Shunxiang; Zheng, Xin; Jiao, Binghua; Wang, Lianghua

    2016-07-01

    Aptamers are functional single-stranded DNA or RNA oligonucleotides, selected in vitro by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which can fold into stable unique three-dimensional structures that bind their target ligands with high affinity and specificity. Although aptamers show a number of favorable advantages such as better stability and easier modification when compared with the properties of antibodies, only a handful of aptamers have entered clinical trials and only one, pegaptanib, has received US Food and Drug Administration approval for clinical use. The main reasons that limit the practical application of aptamers are insufficient nuclease stability, bioavailability, thermal stability, or even affinity. Some aptamers obtained from modified libraries show better properties; however, polymerase amplification of nucleic acids containing non-natural bases is currently a primary drawback of the SELEX process. This review focuses on several post-SELEX optimization strategies of aptamers identified in recent years. We describe four common methods in detail: truncation, chemical modification, bivalent or multivalent aptamer construction, and mutagenesis. We believe that these optimization strategies should improve one or more specific properties of aptamers, and the type of feature(s) selected for improvement will be dependent on the application purpose. PMID:27173394

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. Biostable ssDNA Aptamers Specific for Hodgkin Lymphoma

    OpenAIRE

    Parekh, Parag; Kamble, Sanchit; Zhao, Nianxi; Zeng, Zihua; Wen, Jianguo; Yuan, Bin; Zu, Youli

    2013-01-01

    As a “chemical antibody”, oligonucleotide aptamers can specifically bind to their target molecules. However, clinical potential of aptamers in disease diagnosis is not yet fully explored. Using a tumor cell-based selection protocol, we developed single-stranded DNA aptamers for Hodgkin lymphoma (HL) tumor cells. The aptamers specifically bound to HL cells with a high affinity, reaching maximal cell binding at 10 nM final concentration. Importantly, the aptamers were able to selectively detect...

  8. Targeting cyclin-dependent kinases in Drosophila with peptide aptamers

    OpenAIRE

    Kolonin, Mikhail G.; Finley, Russell L.

    1998-01-01

    Two-hybrid technology provides a simple way to isolate small peptide aptamers that specifically recognize and strongly bind to a protein of interest. These aptamers have the potential to dominantly interfere with specific activities of their target proteins and, therefore, could be used as in vivo inhibitors. Here we explore the ability to use peptide aptamers as in vivo inhibitors by expressing aptamers directed against cell cycle regulators in Drosophila. We expressed two peptide aptamers, ...

  9. DNA Aptamers in the Diagnosis and Treatment of Human Diseases

    OpenAIRE

    Qinchang Zhu; Ge Liu; Masaaki Kai

    2015-01-01

    Aptamers have a promising role in the field of life science and have been extensively researched for application as analytical tools, therapeutic agents and as vehicles for targeted drug delivery. Compared with RNA aptamers, DNA aptamers have inherent advantages in stability and facility of generation and synthesis. To better understand the specific potential of DNA aptamers, an overview of the progress in the generation and application of DNA aptamers in human disease diagnosis and therapy a...

  10. Hi-Fi SELEX: A High-Fidelity Digital-PCR Based Therapeutic Aptamer Discovery Platform.

    Science.gov (United States)

    Ouellet, Eric; Foley, Jonathan H; Conway, Edward M; Haynes, Charles

    2015-08-01

    Current technologies for aptamer discovery typically leverage the systematic evolution of ligands by exponential enrichment (SELEX) concept by recursively panning semi-combinatorial ssDNA or RNA libraries against a molecular target. The expectation is that this iterative selection process will be sufficiently stringent to identify a candidate pool of specific high-affinity aptamers. However, failure of this process to yield promising aptamers is common, due in part to (i) limitations in library designs, (ii) retention of non-specific aptamers during screening rounds, (iii) excessive accumulation of amplification artifacts, and (iv) the use of screening criteria (binding affinity) that does not reflect therapeutic activity. We report a new selection platform, High-Fidelity (Hi-Fi) SELEX, that introduces fixed-region blocking elements to safeguard the functional diversity of the library. The chemistry of the target-display surface and the composition of the equilibration solvent are engineered to strongly inhibit non-specific retention of aptamers. Partition efficiencies approaching 10(6) are thereby realized. Retained members are amplified in Hi-Fi SELEX by digital PCR in a manner that ensures both elimination of amplification artifacts and stoichiometric conversion of amplicons into the single-stranded library required for the next selection round. Improvements to aptamer selections are first demonstrated using human α-thrombin as the target. Three clinical targets (human factors IXa, X, and D) are then subjected to Hi-Fi SELEX. For each, rapid enrichment of ssDNA aptamers offering an order-nM mean equilibrium dissociation constant (Kd) is achieved within three selection rounds, as quantified by a new label-free qPCR assay reported here. Therapeutic candidates against factor D are identified. PMID:25727321

  11. Towards an Upper-Level Ontology for Molecular Biology

    OpenAIRE

    Schulz, Stefan; Beisswanger, Elena; Wermter, Joachim; Hahn, Udo

    2006-01-01

    There is a growing need for the general-purpose description of the basic ontological entities in the life sciences domain. Up until now, upper-level models are mainly purpose-driven, such as the GENIA ontology, originally devised as a vocabulary for corpus annotation. As an alternative, we here present BioTop, a description-logic-based top-level ontology for molecular biology, as an ontologically more conscious re-design of the GENIA ontology.

  12. Towards an upper level ontology for molecular biology.

    Science.gov (United States)

    Schulz, Stefan; Beisswanger, Elena; Wermter, Joachim; Hahn, Udo

    2006-01-01

    There is a growing need for the general-purpose description of the basic conceptual entities in the life sciences. Up until now, upper level models have mainly been purpose-driven, such as the GENIA ontology, originally devised as a vocabulary for corpus annotation. As an alternative,we here present BioTop, a description-logic-based top level ontology for molecular biology, which we consider as an ontologically conscious redesign of the GENIA ontology. PMID:17238430

  13. Chemiluminescence assay for the glycoprotein tenascin-C based on aptamer-modified carboxylated magnetic carbon nanoparticles

    International Nuclear Information System (INIS)

    We report on a method for chemiluminescence (CL) determination of the glycoprotein tenascin-C. Carboxylated carbon nanoparticles (cCNPs) were prepared from activated carbon. Next, the cCNPs were conjugated to magnetic beads (MBs) with a diameter of ∼1 μm by linking its carboxy groups to the amino groups of the MBs. The assay involves the following steps: (a) An aptamer labeled with the CL reagent N-(4-aminobutyl)-N-ethylisoluminol (ABEI) (the labeled aptamer) was adsorbed onto the surface of the carboxy-modified magnetic carbon nanoparticles to form labeled aptamer modified cCNPs-MBs. (b) On addition of sample tenascin-C, it will interact with the labeled aptamer to form a complex with the labeled aptamer. (c) This tenascin-aptamer complex is then dissociated from the surface of the particles and detected by CL whose intensity is linearly related to the concentration of tenascin-C in the 1 pM to 1 nM range. The detection limit is as low as 0.4 pM, and the RSD is 4.2 % at a 50 pM level (for n = 7). The method has been successfully applied to the determination of tenascin-C in human serum samples and holds promise as a widely applicable general platform for aptamer-based CL detection of proteins. (author)

  14. Designing an university-level module on molecular imaging chemistry

    International Nuclear Information System (INIS)

    Full text: Why do we need radiopharmacy, radiopharmacy, radiopharmacy training? In this post-genomic era, molecular imaging has gain tremendous interest not only amongst physicians but also from biologists, chemists, physicists, engineers, statisticians, pharmaceutical companies and even from governments. There is no doubt that nuclear medicine has been engaged in molecular medicine more than one decade ago. Positron emission tomography (PET) has reawaken interest in long forgotten radiopharmacy. Only major hospitals in the developed countries have invested in the development of dedicated radiopharmacy laboratory and training or recruitment of radiopharmacist. But PET has forced nuclear medicine to create a radiopharmacy unit and adopt radiopharmacy guidelines such as good radiopharmaceutical practice (GRPP) and good manufacturing practice (GMP). It is compounded by the fact that SPECT radiopharmaceutical chemistry has advanced significantly for both diagnostics and therapeutics, which calls for a high level of understanding on radiopharmaceutical chemistry and technical know-how. These factors eventually lead to introduction of tran ing program, courses and degree program. The most striking examples will be European Association of Nuclear Medicine (EANM) radiopharmacy courses and a series of IAEA activities on GRPP, GMP and technologist training programs. Various forms of training or education program can be formulated for various levels, starting from basic radiopharmacy course to PhD program, depending on the following factors; (1) National interest and policies on bio/medical sector; (2) Size of the nuclear medicine community in the respective country; (3) Institution interest and policies; and (4) Existing infrastructure and programs. Current Radiopharmacy Education in Singapore: In Singapore, all of the major nuclear medicine centers are supervised by radiopharmacists with PhD degree. All of the nuclear medicine technologists in the major centers have got

  15. Selection of aptamers for metabolite sensing and construction of optical nanosensors

    DEFF Research Database (Denmark)

    Long, Yi; Pfeiffer, Franziska; Mayer, Günter;

    2016-01-01

    -tuned to a desired measuring range in the selection process. Here we first describe the selection of metabolite binding aptamers, how they are transformed into signaling molecules using a molecular beacon construct and then how they are inserted into nanoparticles. Finally, we briefly describe how the sensors...

  16. 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.

  17. 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

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

    International Nuclear Information System (INIS)

    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

  19. Aptamer Affinity Maturation by Resampling and Microarray Selection.

    Science.gov (United States)

    Kinghorn, Andrew B; Dirkzwager, Roderick M; Liang, Shaolin; Cheung, Yee-Wai; Fraser, Lewis A; Shiu, Simon Chi-Chin; Tang, Marco S L; Tanner, Julian A

    2016-07-19

    Aptamers have significant potential as affinity reagents, but better approaches are critically needed to discover higher affinity nucleic acids to widen the scope for their diagnostic, therapeutic, and proteomic application. Here, we report aptamer affinity maturation, a novel aptamer enhancement technique, which combines bioinformatic resampling of aptamer sequence data and microarray selection to navigate the combinatorial chemistry binding landscape. Aptamer affinity maturation is shown to improve aptamer affinity by an order of magnitude in a single round. The novel aptamers exhibited significant adaptation, the complexity of which precludes discovery by other microarray based methods. Honing aptamer sequences using aptamer affinity maturation could help optimize a next generation of nucleic acid affinity reagents. PMID:27346322

  20. Design Strategies for Aptamer-Based Biosensors

    Directory of Open Access Journals (Sweden)

    Kun Han

    2010-05-01

    Full Text Available Aptamers have been widely used as recognition elements for biosensor construction, especially in the detection of proteins or small molecule targets, and regarded as promising alternatives for antibodies in bioassay areas. In this review, we present an overview of reported design strategies for the fabrication of biosensors and classify them into four basic modes: target-induced structure switching mode, sandwich or sandwich-like mode, target-induced dissociation/displacement mode and competitive replacement mode. In view of the unprecedented advantages brought about by aptamers and smart design strategies, aptamer-based biosensors are expected to be one of the most promising devices in bioassay related applications.

  1. Nanoparticles of copper stimulate angiogenesis at systemic and molecular level.

    Science.gov (United States)

    Mroczek-Sosnowska, Natalia; Sawosz, Ewa; Vadalasetty, Krishna Prasad; Łukasiewicz, Monika; Niemiec, Jan; Wierzbicki, Mateusz; Kutwin, Marta; Jaworski, Sławomir; Chwalibog, André

    2015-01-01

    Copper is a key element affecting blood vessel growth and muscle development. However, the ions released from Cu salts are toxic. Given their specific physicochemical properties, nanoparticles of Cu (NanoCu) may have different bioactivity and affect the development of blood vessel and muscles in a different manner than Cu salts. The objective of the study was to evaluate the influence of NanoCu on embryo development and angiogenesis at the systemic and molecular level, in experiments using a chick embryo model. Fertilized chicken eggs were divided into a control group, and groups injected with a placebo, CuSO4 or NanoCu. Embryo development at the whole body level and molecular indices using an embryo chorioallantoic membrane model were measured during embryogenesis. The present study indicated for the first time that NanoCu have pro-angiogenic properties at the systemic level, to a greater degree than CuSO4 salt. The properties of NanoCu were confirmed at the molecular level, demonstrating significant effects on mRNA concentration and on mRNA gene expression of all pro-angiogenic and pro-proliferative genes measured herein. PMID:25741768

  2. Nanoparticles of Copper Stimulate Angiogenesis at Systemic and Molecular Level

    Directory of Open Access Journals (Sweden)

    Natalia Mroczek-Sosnowska

    2015-03-01

    Full Text Available Copper is a key element affecting blood vessel growth and muscle development. However, the ions released from Cu salts are toxic. Given their specific physicochemical properties, nanoparticles of Cu (NanoCu may have different bioactivity and affect the development of blood vessel and muscles in a different manner than Cu salts. The objective of the study was to evaluate the influence of NanoCu on embryo development and angiogenesis at the systemic and molecular level, in experiments using a chick embryo model. Fertilized chicken eggs were divided into a control group, and groups injected with a placebo, CuSO4 or NanoCu. Embryo development at the whole body level and molecular indices using an embryo chorioallantoic membrane model were measured during embryogenesis. The present study indicated for the first time that NanoCu have pro-angiogenic properties at the systemic level, to a greater degree than CuSO4 salt. The properties of NanoCu were confirmed at the molecular level, demonstrating significant effects on mRNA concentration and on mRNA gene expression of all pro-angiogenic and pro-proliferative genes measured herein.

  3. Targeting hepatocellular carcinoma with aptamer-functionalized PLGA/PLA-PEG nanoparticles

    Science.gov (United States)

    Weigum, Shannon E.; Sutton, Melissa; Barnes, Eugenia; Miller, Sarah; Betancourt, Tania

    2014-08-01

    Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, particularly in regions where chronic Hepatitis B and C infections are common. Nanoparticle assemblies that incorporate high-affinity aptamers which specifically bind malignant hepatocellular carcinoma cells could be useful for targeted drug delivery or enhancing contrast with existing ablation therapies. The in vitro interactions of a tumor-specific aptamer, TLS11a, were characterized in a hepatoma cell line via live-cell fluorescence imaging, SDS-PAGE and Western Blotting techniques. Cell surface binding of the aptamer-AlexaFluor®546 conjugate was found to occur within 20 minutes of initial exposure, followed by internalization and localization to late endosomes or lysosomes using a pH-sensitive LysoSensor™ Green dye and confocal microscopy. Aptamer-functionalized polymer nanoparticles containing poly(lactic-co-glycolic acid) (PLGA) and poly(lactide)-b-poly(ethylene glycol) (PLA-PEG) were then prepared by nanoprecipitation and passively loaded with the chemotherapeutic agent, doxorubicin, yielding spherical nanoparticles approximately 50 nm in diameter. Targeted drug delivery and cytotoxicity was assessed using live/dead fluorescent dyes and a MTT colorimetric viability assay with elevated levels of cell death found in cultures treated with either the aptamer-coated and uncoated polymer nanoparticles. Identification and characterization of the cell surface protein epitope(s) recognized by the TLS11a aptamer are ongoing along with nanoparticle optimization, but these preliminary studies support continued investigation of this aptamer and functionalized nanoparticle conjugates for targeted labeling and drug delivery within malignant hepatocellular carcinomas.

  4. Molecular-level investigation on electrochemical interfaces by Raman spectroscopy

    Institute of Scientific and Technical Information of China (English)

    TIAN, Zhong-Qun; REN, Bin

    2000-01-01

    The structure and dynamics of electrode/liquid interfaces play an increasingly important role in electrochemistry. Raman spectroscopy is capable of providing detailed structural information at molecular level and new insight into the interfacial structure, adsorption, reaction, electrocatalysis and corrosion. In this account we will summarize some progresses of surface Raman spectroscopy in the study of electrochemical interfaces, mainly based on our group's work, laying emphasis on the detection sensitivity, spectral resolution, time resolution and spatial resolution as well as the hyphenated technique.

  5. From selection hits to clinical leads: progress in aptamer discovery

    OpenAIRE

    Maier, Keith E.; Levy, Matthew

    2016-01-01

    Aptamers were discovered more than 25 years ago, yet only one has been approved by the US Food and Drug Administration to date. With some noteworthy advances in their chemical design and the enzymes we use to make them, aptamers and aptamer-based therapeutics have seen a resurgence in interest. New aptamer drugs are being approved for clinical evaluation, and it is certain that we will see increasingly more aptamers and aptamer-like drugs in the future. In this review, we will discuss the pro...

  6. Antagonistic effect of disulfide-rich peptide aptamers selected by cDNA display on interleukin-6-dependent cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Nemoto, Naoto, E-mail: nemoto@fms.saitama-u.ac.jp [Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Innovation Center for Startups, National Institute of Advanced Industrial Science and Technology, 2-2-2 Marunouchi, Chiyoda-ku, Tokyo 100-0005 (Japan); Janusys Corporation, 508, Saitama Industrial Technology Center, Skip City, 3-12-18 Kami-Aoki, Kawaguchi, Saitama 333-0844 (Japan); Tsutsui, Chihiro [Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Yamaguchi, Junichi [Innovation Center for Startups, National Institute of Advanced Industrial Science and Technology, 2-2-2 Marunouchi, Chiyoda-ku, Tokyo 100-0005 (Japan); Applied Gene Technology, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Ueno, Shingo [Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan); Machida, Masayuki [Applied Gene Technology, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Kobayashi, Toshikatsu [Innovation Center for Startups, National Institute of Advanced Industrial Science and Technology, 2-2-2 Marunouchi, Chiyoda-ku, Tokyo 100-0005 (Japan); Janusys Corporation, 508, Saitama Industrial Technology Center, Skip City, 3-12-18 Kami-Aoki, Kawaguchi, Saitama 333-0844 (Japan); Sakai, Takafumi [Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570 (Japan)

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer Disulfide-rich peptide aptamer inhibits IL-6-dependent cell proliferation. Black-Right-Pointing-Pointer Disulfide bond of peptide aptamer is essential for its affinity to IL-6R. Black-Right-Pointing-Pointer Inhibitory effect of peptide depends on number and pattern of its disulfide bonds. -- Abstract: Several engineered protein scaffolds have been developed recently to circumvent particular disadvantages of antibodies such as their large size and complex composition, low stability, and high production costs. We previously identified peptide aptamers containing one or two disulfide-bonds as an alternative ligand to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in length) were screened from a random peptide library by in vitro peptide selection using the evolutionary molecular engineering method 'cDNA display'. In this report, the antagonistic activity of the peptide aptamers were examined by an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an IL-6-dependent cell proliferation assay. The results revealed that a disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with similar efficacy to an anti-IL-6R monoclonal antibody.

  7. Characterization of 2 '-fluoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion

    OpenAIRE

    Rhie, A.; Kirby, L.; Sayer, N.; Wellesley, R.; Disterer, P; Sylvester, I; Gill, A; Hope, J.; James, W.; Tahiri-Alaoui, A

    2003-01-01

    We have isolated artificial ligands or aptamers for infectious prions in order to investigate conformational aspects of prion pathogenesis. The aptamers are 2'-fluoro-modified RNA produced by in vitro selection from a large, randomized library. One of these ligands (aptamer SAF-93) had more than 10-fold higher affinity for PrPSc than for recombinant PrPC and inhibited the accumulation of PrPres in near physiological cell-free conversion assay. To understand the molecular basis of these proper...

  8. Aptamers : The New Frontier In Drug Development?

    OpenAIRE

    CARLSON, BOB

    2007-01-01

    Two companies – one focused on diagnostics, the other on therapeutics – want you to know. Their collaborations with some of pharma’s biggest names are making aptamers a force to be reckoned with in the biologic arsenal.

  9. 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.

  10. Small-molecule-dependent split aptamer ligation.

    Science.gov (United States)

    Sharma, Ashwani K; Heemstra, Jennifer M

    2011-08-17

    Here we describe the first use of small-molecule binding to direct a chemical reaction between two nucleic acid strands. The reported reaction is a ligation between two fragments of a DNA split aptamer using strain-promoted azide-alkyne cycloaddition. Utilizing the split aptamer for cocaine, we demonstrate small-molecule-dependent ligation that is dose-dependent over a wide range of cocaine concentrations and is compatible with complex biological fluids such as human blood serum. Moreover, studies of split aptamer ligation at varying salt concentrations and using structurally similar analogues of cocaine have revealed new insight into the assembly and small-molecule binding properties of the cocaine split aptamer. The ability to translate the presence of a small-molecule target into the output of DNA ligation is anticipated to enable the development of new, broadly applicable small-molecule detection assays. PMID:21761903

  11. Multi-level molecular modelling for plasma medicine

    International Nuclear Information System (INIS)

    Modelling at the molecular or atomic scale can be very useful for obtaining a better insight in plasma medicine. This paper gives an overview of different atomic/molecular scale modelling approaches that can be used to study the direct interaction of plasma species with biomolecules or the consequences of these interactions for the biomolecules on a somewhat longer time-scale. These approaches include density functional theory (DFT), density functional based tight binding (DFTB), classical reactive and non-reactive molecular dynamics (MD) and united-atom or coarse-grained MD, as well as hybrid quantum mechanics/molecular mechanics (QM/MM) methods. Specific examples will be given for three important types of biomolecules, present in human cells, i.e. proteins, DNA and phospholipids found in the cell membrane. The results show that each of these modelling approaches has its specific strengths and limitations, and is particularly useful for certain applications. A multi-level approach is therefore most suitable for obtaining a global picture of the plasma–biomolecule interactions. (paper)

  12. Multi-level molecular modelling for plasma medicine

    Science.gov (United States)

    Bogaerts, Annemie; Khosravian, Narjes; Van der Paal, Jonas; Verlackt, Christof C. W.; Yusupov, Maksudbek; Kamaraj, Balu; Neyts, Erik C.

    2016-02-01

    Modelling at the molecular or atomic scale can be very useful for obtaining a better insight in plasma medicine. This paper gives an overview of different atomic/molecular scale modelling approaches that can be used to study the direct interaction of plasma species with biomolecules or the consequences of these interactions for the biomolecules on a somewhat longer time-scale. These approaches include density functional theory (DFT), density functional based tight binding (DFTB), classical reactive and non-reactive molecular dynamics (MD) and united-atom or coarse-grained MD, as well as hybrid quantum mechanics/molecular mechanics (QM/MM) methods. Specific examples will be given for three important types of biomolecules, present in human cells, i.e. proteins, DNA and phospholipids found in the cell membrane. The results show that each of these modelling approaches has its specific strengths and limitations, and is particularly useful for certain applications. A multi-level approach is therefore most suitable for obtaining a global picture of the plasma-biomolecule interactions.

  13. DNA aptamers detecting generic amyloid epitopes

    OpenAIRE

    Mitkevich, Olga V.; Kochneva-Pervukhova, Natalia V; Surina, Elizaveta R.; Benevolensky, Sergei V.; Kushnirov, Vitaly V.; Ter-Avanesyan, Michael D.

    2012-01-01

    Amyloids are fibrillar protein aggregates resulting from non-covalent autocatalytic polymerization of various structurally and functionally unrelated proteins. Previously we have selected DNA aptamers, which bind specifically to the in vitro assembled amyloid fibrils of the yeast prionogenic protein Sup35. Here we show that such DNA aptamers can be used to detect SDS-insoluble amyloid aggregates of the Sup35 protein, and of some other amyloidogenic proteins, including mouse PrP, formed in yea...

  14. DNA-Aptamers Binding Aminoglycoside Antibiotics

    OpenAIRE

    Nadia Nikolaus; Beate Strehlitz

    2014-01-01

    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 aminog...

  15. Direct Selection of RNA Beacon Aptamers

    OpenAIRE

    Morse, Daniel P.

    2007-01-01

    A method for the direct selection of RNA molecules that can be easily converted into beacon aptamers is presented. Beacon aptamers are fluorescently labeled nucleic acids that signal the presence of a specific ligand through changes in fluorescence intensity. Typically, ligand binding causes an increase in fluorescence intensity by inducing a conformational change that separates a fluorophore/quencher pair. The method presented here simultaneously selects for ligand binding and induction of a...

  16. RAPID-SELEX for RNA Aptamers

    OpenAIRE

    Szeto, Kylan; Latulippe, David R.; Ozer, Abdullah; Pagano, John M.; Brian S White; Shalloway, David; Lis, John T.; Craighead, Harold G.

    2013-01-01

    Aptamers are high-affinity ligands selected from DNA or RNA libraries via SELEX, a repetitive in vitro process of sequential selection and amplification steps. RNA SELEX is more complicated than DNA SELEX because of the additional transcription and reverse transcription steps. Here, we report a new selection scheme, RAPID-SELEX (RNA Aptamer Isolation via Dual-cycles SELEX), that simplifies this process by systematically skipping unnecessary amplification steps. Using affinity microcolumns, we...

  17. Aptamer Based Microsphere Biosensor for Thrombin Detection

    OpenAIRE

    Xudong Fan; White, Ian M.; Suter, Jonathan D.; Hongying Zhu

    2006-01-01

    We have developed an optical microsphere resonator biosensor using aptamer as receptor for the measurement of the important biomolecule thrombin. The sphere surface is modified with anti-thrombin aptamer, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the sphere surface is monitored by the spectral position of the microsphere's whispering gallery mode resonances. A detection limit on the order of 1 NIH Unit/mL is demonstrated. Control experiments...

  18. Aptamers in oncology: a diagnostic perspective

    OpenAIRE

    Khan, Huma; Missailidis, Sotiris

    2008-01-01

    Nucleic acid sequences can produce a wide variety of three-dimensional conformations. Some of these structural forms are able to interact with proteins and small molecules with high affinity and specificity. These sequences, comprising either double or single stranded oligonucleotides, are called 'aptamers' based on the Greek word aptus, which means 'to fit'. Using an efficient selection process, randomised oligonucleotide libraries can be rapidly screened for aptamers with the appropriate bi...

  19. Aptamers: A Feasible Technology in Cancer Immunotherapy

    Science.gov (United States)

    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. PMID:27413756

  20. Structural computational modeling of RNA aptamers.

    Science.gov (United States)

    Xu, Xiaojun; Dickey, David D; Chen, Shi-Jie; Giangrande, Paloma H

    2016-07-01

    RNA aptamers represent an emerging class of biologics that can be easily adapted for personalized and precision medicine. Several therapeutic aptamers with desirable binding and functional properties have been developed and evaluated in preclinical studies over the past 25years. However, for the majority of these aptamers, their clinical potential has yet to be realized. A significant hurdle to the clinical adoption of this novel class of biologicals is the limited information on their secondary and tertiary structure. Knowledge of the RNA's structure would greatly facilitate and expedite the post-selection optimization steps required for translation, including truncation (to reduce costs of manufacturing), chemical modification (to enhance stability and improve safety) and chemical conjugation (to improve drug properties for combinatorial therapy). Here we describe a structural computational modeling methodology that when coupled to a standard functional assay, can be used to determine key sequence and structural motifs of an RNA aptamer. We applied this methodology to enable the truncation of an aptamer to prostate specific membrane antigen (PSMA) with great potential for targeted therapy that had failed previous truncation attempts. This methodology can be easily applied to optimize other aptamers with therapeutic potential. PMID:26972787

  1. Sensitive analytical performance of folding based biosensor using methylene blue tagged aptamers.

    Science.gov (United States)

    Catanante, Gaëlle; Mishra, Rupesh K; Hayat, Akhtar; Marty, Jean-Louis

    2016-06-01

    This work demonstrates the development of a folding based electrochemical aptasensor using methylene blue (MB) tagged anti-Ochratoxin A (OTA) aptamers. Different aptamer coupling strategies were tested using Hexamethylenediamine, polyethylene glycol, simple adsorption and diazonium coupling mechanism. The best sensitivity was recorded by oxidation of amines using hexamethylenediamine (HDMA) on screen printed carbon electrode (SPCE). To achieve the direct detection of OTA, aptamer conjugated redox probe was used and detection was demonstrated based on the conformational changes in aptamer structure upon OTA sensing. Signaling in this class of sensors arises from changes in electron transfer efficiency upon target-induced changes in the conformation/flexibility of the aptamer probe. These changes can be readily recorded electrochemically. The developed aptasensor is unique in its own mechanism as redox probe tagged aptamer coupling such as MB has never been tried to immobilize using long carbon chain spacers as, addition of spacers would provide more sensitive detection methods. A good dynamic range 0.01-5ng/ml was obtained for OTA with Limit of detection (LOD) 0.01ng/ml and Limit of quantification (LOQ) of 0.03ng/ml respectively. The good reproducibility was recorded with RSD% of 3.75. The obtained straight line equation was y=0.4035x+0.90311, r=0.9976. We believe that the sensor design guidelines outlined here represents a general strategy for developing new folding-based electrochemical aptasensors. The developed aptasensor was extended to screen cocoa samples for OTA contamination. The cocoa samples were extracted and purified using molecular imprinted polymer (MIP) columns. The aptasensor displayed good recovery values in the range 84-85% thus, exhibited the effectiveness of proposed aptasensor for such complex matrices. PMID:27130100

  2. Soy protein isolate molecular level contributions to bulk adhesive properties

    Science.gov (United States)

    Shera, Jeanne Norton

    Increasing environmental awareness and the recognized health hazards of formaldehyde-based resins has prompted a strong demand for environmentally-responsible adhesives for wood composites. Soy protein-based adhesives have been shown to be commercially viable with 90-day shelf stability and composite physical properties comparable to those of commercial formaldehyde-based particleboards. The main research focus is to isolate and characterize the molecular level features in soy protein isolate responsible for providing mechanical properties, storage stability, and water resistance during adhesive formulation, processing, and wood composite fabrication. Commercial composite board will be reviewed to enhance our understanding of the individual components and processes required for particleboard production. The levels of protein structure will be defined and an overview of current bio-based technology will be presented. In the process, the logic for utilizing soy protein as a sole binder in the adhesive will be reinforced. Variables such as adhesive components, pH, divalent ions, blend aging, protein molecular weight, formulation solids content, and soy protein functionalization will relate the bulk properties of soy protein adhesives to the molecular configuration of the soybean protein. This work has demonstrated that when intermolecular beta-sheet interactions and protein long-range order is disrupted, viscosity and mechanical properties decrease. Storage stability can be maintained through the stabilization of intermolecular beta-sheet interactions. When molecular weight is reduced through enzymatic digestion, long-range order is disrupted and viscosity and mechanical properties decrease accordingly. Processibility and physical properties must be balanced to increase solids while maintaining low viscosity, desirable mechanical properties, and adequate storage stability. The structure of the soybean protein must be related to the particleboard bulk mechanical

  3. A nanoresonant gold-aptamer probe for rapid and sensitive detection of thrombin

    International Nuclear Information System (INIS)

    Resonance light scattering (RLS) is a sensitive technique for monitoring scattered light induced by extended aggregates of chromophores. It has been widely used to study aggregations for its simple manipulation, high sensitivity and great versatility. Gold nanoparticles generate colorful light-scattering signals due to their unique surface plasmon resonances, hence extraordinary light scattering upon aggregation. In this paper we report a rapid and sensitive method based on gold nanoparticles and DNA aptamer to detect protein biomarkers by RLS. Thiol modified thrombin aptamer was covalently assembled to the surface of gold nanoparticles as nanobio probes. As thrombin has two specific binding sites for its aptamer, it can bridge the well dispersed nanoparticles and lead to a network of particle aggregations. The formation of aggregation ia measured by RLS, and the specific detection of thrombin at nM level is achieved. The method has good specificity. (authors)

  4. 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...

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

    OpenAIRE

    Baby Santosh; Pramod K. Yadava

    2014-01-01

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

  6. A systematic approach to evolve aptamers with new specificities

    Science.gov (United States)

    Aptamers are single-stranded nucleic acids with high affinities and specificities for the targets against which they are selected. Both features, along with an ability to be integrated into a large variety of sensors, make possible a wide-range of aptamer applications. However, changing aptamer sp...

  7. Graphene oxide-assisted non-immobilized SELEX of okdaic acid aptamer and the analytical application of aptasensor

    Science.gov (United States)

    Gu, Huajie; Duan, Nuo; Wu, Shijia; Hao, Liling; Xia, Yu; Ma, Xiaoyuan; Wang, Zhouping

    2016-02-01

    Okadaic acid (OA) is a low-molecular-weight marine toxin from shellfish that causes abdominal pain, vomiting and diarrhea, i.e., diarrheic shellfish poisoning. In this study, a ssDNA aptamer that specifically binds to OA with high affinity was obtained via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assisted by graphene oxide (GO). This aptamer was then applied to fabricate a novel direct competitive enzyme-linked aptamer assay (ELAA). At the optimized conditions, this ELAA method showed a low detection limit (LOD of 0.01 ng/mL), wide linear range (from 0.025 to 10 ng/mL), good recovery rate (92.86-103.34% in OA-spiked clam samples) and repeatability (RSD of 2.28-4.53%). The proposed method can be used to detect OA in seafood products with high sensitivity and can potentially be adapted for the determination of other small molecular analytes.

  8. The novel amyloid-beta peptide aptamer inhibits intracellular amyloid-beta peptide toxicity

    Institute of Scientific and Technical Information of China (English)

    Xu Wang; Yi Yang; Mingyue Jia; Chi Ma; Mingyu Wang; Lihe Che; Yu Yang; Jiang Wu

    2013-01-01

    Amyloid β peptide binding alcohol dehydrogenase (ABAD) decoy peptide (DP) can competitively antagonize binding of amyloid β peptide to ABAD and inhibit the cytotoxic effects of amyloid β peptide. Based on peptide aptamers, the present study inserted ABAD-DP into the disulfide bond of human thioredoxin (TRX) using molecular cloning technique to construct a fusion gene that can express the TRX1-ABAD-DP-TRX2 aptamer. Moreover, adeno-associated virus was used to allow its stable expression. Immunofluorescent staining revealed the co-expression of the transduced fusion gene TRX1-ABAD-DP-TRX2 and amyloid β peptide in NIH-3T3 cells, indicating that the TRX1-ABAD-DP-TRX2 aptamer can bind amyloid β peptide within cells. In addition, cell morphology and MTT results suggested that TRX1-ABAD-DP-TRX2 attenuated amyloid β peptide-induced SH-SY5Y cell injury and improved cell viability. These findings confirmed the possibility of constructing TRX-based peptide aptamer using ABAD-DP. Moreover, TRX1-ABAD-DP-TRX2 inhibited the cytotoxic effect of amyloid β peptide.

  9. An RNA Aptamer Targets the PDZ-Binding Motif of the HPV16 E6 Oncoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Belyaeva, Tamara A.; Nicol, Clare; Cesur, Özlem [School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom); Travé, Gilles [UMR 7242 CNRS-Université de Strasbourg, Ecole Supérieure de Biotechnologie, Boulevard Sébastien Brant, Illkirch 67412 (France); Blair, George Eric; Stonehouse, Nicola J., E-mail: n.j.stonehouse@leeds.ac.uk [School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom)

    2014-07-24

    Human papillomavirus 16 (HPV16) is a high-risk DNA tumour virus which is the primary causative agent of cervical cancer. Cell transformation arises from deregulated expression of the E6 and E7 oncogenes. E6 has been shown to bind a number of cellular proteins, including p53 and proteins containing a PDZ domain. This study reports the first RNA aptamers to E6. These have been employed as molecular tools to further investigate E6-p53 and E6-PDZ interactions. This study is focussed on two aptamers (termed F2 and F4) which induced apoptosis in cells derived from an HPV16-transformed cervical carcinoma. The molecules were able to inhibit the interaction between E6 and PDZ1 from Magi1, with F2 being the most effective inhibitor. Neither of the aptamers inhibited E6-p53 interaction or p53 degradation. This study shows the specificity of this approach and highlights the potential benefits of the E6 aptamers as potential therapeutic or diagnostic agents in the future.

  10. 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. PMID:26078174

  11. An RNA Aptamer Targets the PDZ-Binding Motif of the HPV16 E6 Oncoprotein

    International Nuclear Information System (INIS)

    Human papillomavirus 16 (HPV16) is a high-risk DNA tumour virus which is the primary causative agent of cervical cancer. Cell transformation arises from deregulated expression of the E6 and E7 oncogenes. E6 has been shown to bind a number of cellular proteins, including p53 and proteins containing a PDZ domain. This study reports the first RNA aptamers to E6. These have been employed as molecular tools to further investigate E6-p53 and E6-PDZ interactions. This study is focussed on two aptamers (termed F2 and F4) which induced apoptosis in cells derived from an HPV16-transformed cervical carcinoma. The molecules were able to inhibit the interaction between E6 and PDZ1 from Magi1, with F2 being the most effective inhibitor. Neither of the aptamers inhibited E6-p53 interaction or p53 degradation. This study shows the specificity of this approach and highlights the potential benefits of the E6 aptamers as potential therapeutic or diagnostic agents in the future

  12. Expected and unexpected features of protein-binding RNA aptamers

    DEFF Research Database (Denmark)

    Bjerregaard, Nils; Andreasen, Peter A; Dupont, Daniel M

    2016-01-01

    RNA molecules with high affinity to specific proteins can be isolated from libraries of up to 10(16) different RNA sequences by systematic evolution of ligands by exponential enrichment (SELEX). These so-called protein-binding RNA aptamers are often interesting, e.g., as modulators of protein...... function for therapeutic use, for probing the conformations of proteins, for studies of basic aspects of nucleic acid-protein interactions, etc. Studies on the interactions between RNA aptamers and proteins display a number of expected and unexpected features, including the chemical nature of the...... interacting RNA-protein surfaces, the conformation of protein-bound aptamer versus free aptamer, the conformation of aptamer-bound protein versus free protein, and the effects of aptamers on protein function. Here, we review current insights into the details of RNA aptamer-protein interactions. For further...

  13. Modulating the Substrate Selectivity of DNA Aptamers Using Surfactants.

    Science.gov (United States)

    Peterson, Amberlyn M; Jahnke, Frank M; Heemstra, Jennifer M

    2015-11-01

    Nucleic acid aptamers have a number of advantages compared to antibodies, including greater ease of production and increased thermal stability. We hypothesized that aptamers may also be capable of functioning in the presence of high concentrations of surfactants, which readily denature antibodies and other protein-based affinity reagents. Here we report the first systematic investigation into the compatibility of DNA aptamers with surfactants. We find that neutral and anionic surfactants have only a minor impact on the ability of aptamers to fold and bind hydrophilic target molecules. Additionally, we demonstrate that surfactants can be utilized to modulate the substrate binding preferences of aptamers, likely due to the sequestration of hydrophobic target molecules within micelles. The compatibility of aptamers with commonly used surfactants is anticipated to expand their scope of potential applications, and the ability to modulate the substrate binding preferences of aptamers using a simple additive provides a novel route to increasing their selectivity in analytical applications. PMID:26465173

  14. tPA-binding RNA Aptamers

    DEFF Research Database (Denmark)

    Bjerregaard, Nils

    2015-01-01

    -density lipoprotein receptor Related Protein-1 (LRP-1). Here, we describe the selection and characterisation of structured RNA ligands (“RNA aptamers”) to tPA, K18 and K32. Both aptamers were truncated to minimal 32-nucleotide constructs (v2) with improved or unchanged activities, and were shown to bind tPA with low...... 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......, and upon conjugation to serum albumin. K18v2 was able to inhibit tPA-induced fibrinogen depletion in vitro, which may provide additional benefits in stroke treatment. A conjugate of both aptamers separated by a linker encompassed the activities of both constituent sequences, and additionally possessed...

  15. 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.

  16. Nucleic Acid Aptamers for Living Cell Analysis

    Science.gov (United States)

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

    2014-06-01

    Cells as the building blocks of life determine the basic functions and properties of a living organism. Understanding the structure and components of a cell aids in the elucidation of its biological functions. Moreover, knowledge of the similarities and differences between diseased and healthy cells is essential to understanding pathological mechanisms, identifying diagnostic markers, and designing therapeutic molecules. However, monitoring the structures and activities of a living cell remains a challenging task in bioanalytical and life science research. To meet the requirements of this task, aptamers, as “chemical antibodies,” have become increasingly powerful tools for cellular analysis. This article reviews recent advances in the development of nucleic acid aptamers in the areas of cell membrane analysis, cell detection and isolation, real-time monitoring of cell secretion, and intracellular delivery and analysis with living cell models. Limitations of aptamers and possible solutions are also discussed.

  17. Improvement in the drug delivery and anti-tumor efficacy of PEGylated liposomal doxorubicin by targeting RNA aptamers in mice bearing breast tumor model.

    Science.gov (United States)

    Moosavian, Seyedeh Alia; Abnous, Khalil; Badiee, Ali; Jaafari, Mahmoud Reza

    2016-03-01

    Targeted delivery by ligands such as aptamers, is a promising method to increase the efficiency of PEGylated-liposomal doxorubicin (PL-Dox). In this study, we have successfully conjugated our recently developed anti-breast cancer RNA aptamer (TSA14) to the surface of PL-Dox and characterized for their size, zeta potential, Dox percent encapsulation and release properties in the presence of fetal bovine serum. In vitro experiments showed that aptamer could improve cellular uptake and cytotoxicity of PL-Dox in TUBO breast cell line. In mice bearing TUBO breast tumor, although, the doxorubicin plasma level of liposomal doxorubicin did not significantly change after modification of nanoparticles with aptamer, however, much higher tumor accumulation of Dox as compared with non-targeted liposomes proved the tumor-targeting capability of aptamers. In the same way, aptamer-PL-Dox improved anti-tumor efficiency of liposomes in TUBO breast tumor in mice compared to non-targeted liposomes. Overall, the results showed that aptamer decoration of PL-Dox could significantly improve selectivity and the therapeutic efficacy of liposomal DOX and merits further investigation. PMID:26722819

  18. Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold.

    Science.gov (United States)

    Bruno, John G

    2014-01-01

    Preliminary studies aimed at improving the sensitivity of foodborne pathogen detection via lateral flow (LF) test strips by use of high affinity DNA aptamers for capture and reporter functions when coupled to red-emitting quantum dots (Qdot 655) are reported. A variety of DNA aptamers developed against Escherichia coli, Listeria monocytogenes, and Salmonella enterica were paired in capture and reporter combinations to determine which yielded the strongest detection of their cognate bacteria using a colloidal gold screening system. Several promising sandwich combinations were identified for each of the three bacterial LF strip systems. The best E. coli aptamer-LF system was further studied and yielded a visible limit of detection (LOD) of ~3,000 E. coli 8739 and ~6,000 E. coli O157:H7 in buffer. These LODs were reduced to ~300-600 bacterial cells per test respectively by switching to a Qdot 655 aptamer-LF system. Novel aspects of these assays such as the use of high levels of detergents to avoid quantum dot agglutination and enhance migration in analytical membranes, identification of optimal analytical membrane types, UV-immobilization of capture aptamers, and novel dual biotin/digoxigenin-end labeled aptamer streptavidin-colloidal gold or -Qdot 655 conjugates plus anti-digoxigenin antibody control lines are also discussed. In general, this work provides proof-of-principle for highly sensitive aptamer-Qdot LF strip assays for rapid foodborne pathogen detection. PMID:25437803

  19. Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold

    Directory of Open Access Journals (Sweden)

    John G. Bruno

    2014-04-01

    Full Text Available Preliminary studies aimed at improving the sensitivity of foodborne pathogen detection via lateral flow (LF test strips by use of high affinity DNA aptamers for capture and reporter functions when coupled to red-emitting quantum dots (Qdot 655 are reported. A variety of DNA aptamers developed against Escherichia coli, Listeria monocytogenes, and Salmonella enterica were paired in capture and reporter combinations to determine which yielded the strongest detection of their cognate bacteria using a colloidal gold screening system. Several promising sandwich combinations were identified for each of the three bacterial LF strip systems. The best E. coli aptamer-LF system was further studied and yielded a visible limit of detection (LOD of ~3,000 E. coli 8739 and ~6,000 E. coli O157:H7 in buffer. These LODs were reduced to ~300–600 bacterial cells per test respectively by switching to a Qdot 655 aptamer-LF system. Novel aspects of these assays such as the use of high levels of detergents to avoid quantum dot agglutination and enhance migration in analytical membranes, identification of optimal analytical membrane types, UV-immobilization of capture aptamers, and novel dual biotin/digoxigenin-end labeled aptamer streptavidin-colloidal gold or -Qdot 655 conjugates plus anti-digoxigenin antibody control lines are also discussed. In general, this work provides proof-of-principle for highly sensitive aptamer-Qdot LF strip assays for rapid foodborne pathogen detection.

  20. Gold Nanoparticles Surface Plasmon Resonance Enhanced Signal for the Detection of Small Molecules on Split-Aptamer Microarrays (Small Molecules Detection from Split-Aptamers

    Directory of Open Access Journals (Sweden)

    Feriel Melaine

    2015-02-01

    Full Text Available The detection of small molecules by biosensors remains a challenge for diagnostics in many areas like pharmacology, environment or homeland security. The main difficulty comes from both the low molecular weight and low concentrations of most targets, which generally requires an indirect detection with an amplification or a sandwich procedure. In this study, we combine both strategies as the amplification of Surface Plasmon Resonance imaging (SPRi signal is obtained by the use of gold nanoparticles and the sequence engineering of split-aptamers, short oligonucleotides strands with strong affinity towards small targets, allows for a sandwich structure. Combining those two strategies, we obtained state-of-the-art results in the limit of detection (LOD = 50 nM with the model target adenosine. Furthermore, the SPRi detection led on aptamer microarrays paves the way for potential multi-target detections thanks to the multi-probe imaging approach.

  1. RNA Fluorescence with Light-Up Aptamers

    Science.gov (United States)

    Ouellet, Jonathan

    2016-01-01

    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. PMID:27446908

  2. Identification of malaria parasite-infected red blood cell surface aptamers by inertial microfluidic SELEX (I-SELEX)

    Science.gov (United States)

    Birch, Christina M.; Hou, Han Wei; Han, Jongyoon; Niles, Jacquin C.

    2015-07-01

    Plasmodium falciparum malaria parasites invade and remodel human red blood cells (RBCs) by trafficking parasite-synthesized proteins to the RBC surface. While these proteins mediate interactions with host cells that contribute to disease pathogenesis, the infected RBC surface proteome remains poorly characterized. Here we use a novel strategy (I-SELEX) to discover high affinity aptamers that selectively recognize distinct epitopes uniquely present on parasite-infected RBCs. Based on inertial focusing in spiral microfluidic channels, I-SELEX enables stringent partitioning of cells (efficiency ≥ 106) from unbound oligonucleotides at high volume throughput (~2 × 106 cells min-1). Using an RBC model displaying a single, non-native antigen and live malaria parasite-infected RBCs as targets, we establish suitability of this strategy for de novo aptamer selections. We demonstrate recovery of a diverse set of aptamers that recognize distinct, surface-displayed epitopes on parasite-infected RBCs with nanomolar affinity, including an aptamer against the protein responsible for placental sequestration, var2CSA. These findings validate I-SELEX as a broadly applicable aptamer discovery platform that enables identification of new reagents for mapping the parasite-infected RBC surface proteome at higher molecular resolution to potentially contribute to malaria diagnostics, therapeutics and vaccine efforts.

  3. Orbital Energy Levels in Molecular Hydrogen. A Simple Approach.

    Science.gov (United States)

    Willis, Christopher J.

    1988-01-01

    Described are the energetics involved in the formation of molecular hydrogen using concepts that should be familiar to students beginning the study of molecular orbital theory. Emphasized are experimental data on ionization energies. Included are two-electron atomic and molecular systems. (CW)

  4. Modelling Molecular Mechanisms: A Framework of Scientific Reasoning to Construct Molecular-Level Explanations for Cellular Behaviour

    Science.gov (United States)

    van Mil, Marc H. W.; Boerwinkel, Dirk Jan; Waarlo, Arend Jan

    2013-01-01

    Although molecular-level details are part of the upper-secondary biology curriculum in most countries, many studies report that students fail to connect molecular knowledge to phenomena at the level of cells, organs and organisms. Recent studies suggest that students lack a framework to reason about complex systems to make this connection. In this…

  5. Modelling molecular mechanisms: a framework of scientific reasoning to construct molecular-level explanations for cellular behaviour

    NARCIS (Netherlands)

    van Mil, M.H.W.; Boerwinkel, D.J.; Waarlo, A.J.

    2013-01-01

    Although molecular-level details are part of the upper-secondary biology curriculum in most countries, many studies report that students fail to connect molecular knowledge to phenomena at the level of cells, organs and organisms. Recent studies suggest that students lack a framework to reason about

  6. A tenascin-C aptamer identified by tumor cell SELEX: Systematic evolution of ligands by exponential enrichment

    Science.gov (United States)

    Daniels, Dion A.; Chen, Hang; Hicke, Brian J.; Swiderek, Kristine M.; Gold, Larry

    2003-01-01

    The targeting of molecular repertoires to complex systems rather than biochemically pure entities is an accessible approach that can identify proteins of biological interest. We have probed antigens presented by a monolayer of tumor cells for their ability to interact with a pool of aptamers. A glioblastoma-derived cell line, U251, was used as the target for systematic evolution of ligands by exponential enrichment by using a single-stranded DNA library. We isolated specifically interacting oligonucleotides, and biochemical strategies were used to identify the protein target for one of the aptamers. Here we characterize the interaction of the DNA aptamer, GBI-10, with tenascin-C, an extracellular protein found in the tumor matrix. Tenascin-C is believed to be involved in both embryogenesis and oncogenesis pathways. Systematic evolution of ligands by exponential enrichment appears to be a successful strategy for the a priori identification of targets of biological interest within complex systems. PMID:14676325

  7. Colorimetric detection with aptamer-gold nanoparticle conjugates: effect of aptamer length on response

    Energy Technology Data Exchange (ETDEWEB)

    Chavez, Jorge L. [Wright-Patterson Air Force Base, 711th Human Performance Wing, Human Effectiveness Directorate, Air Force Research Laboratory (United States); MacCuspie, Robert I. [National Institute of Standards and Technology, Ceramics Division (United States); Stone, Morley O.; Kelley-Loughnane, Nancy, E-mail: Nancy.Kelley-Loughnane@wpafb.af.mil [Wright-Patterson Air Force Base, 711th Human Performance Wing, Human Effectiveness Directorate, Air Force Research Laboratory (United States)

    2012-10-15

    A riboflavin binding aptamer (RBA) was used in combination with gold nanoparticles (AuNPs) to detect riboflavin in vitro. The RBA-AuNP conjugates (RBA-AuNPs) responded colorimetrically to the presence of riboflavin and this response could be followed by the naked eye. This system was used as a model to study how modifications on the aptamer sequence affect the RBA-AuNPs' stability and their response to their target. To mimic primers and other sequence modifications typically used in aptamer work, the RBA was extended by adding extra bases to its 5 Prime end. These extra bases were designed to avoid interactions with the RBA binding site. The response of these RBA-AuNPs was evaluated and compared. Dynamic light scattering and UV-aggregation kinetics studies showed that the length of the aptamer significantly affected the RBA-AuNPs' stability and, as a consequence, the magnitude of the detection response to riboflavin. The addition of thymine nucleotides instead of random tails to the RBA showed that the effects observed were not specific to the sequence used. This study shows that modifications of the aptamer sequence provide a means to improve the stability of aptamer-AuNPs conjugates and their sensing response.

  8. Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

    OpenAIRE

    Ausländer, Simon; Fuchs, David; Hürlemann, Samuel; Ausländer, David; Fussenegger, Martin

    2016-01-01

    Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA archit...

  9. Translation and Clinical Development of Antithrombotic Aptamers.

    Science.gov (United States)

    Nimjee, Shahid M; Povsic, Thomas J; Sullenger, Bruce A; Becker, Richard C

    2016-06-01

    Thrombosis is a necessary physiological process to protect the body from uncontrolled bleeding. Pathological thrombus formation can lead to devastating clinical events including heart attack, stroke, deep vein thrombosis, pulmonary embolism, and disseminated intravascular coagulation. Numerous drugs have been developed to inhibit thrombosis. These have been targeted to coagulation factors along with proteins and receptors that activate platelets. While these drugs are effective at preventing blood clotting, their major side effect is inadvertent hemorrhage that can result in significant morbidity and mortality. There exists a need for anticoagulants that are not only effective at preventing thrombosis but can also be readily reversed. Aptamers offer a potential solution, representing a new class of drug agents that can be isolated to any protein and where antidote oligonucleotides can be designed based on the sequence of the aptamer. We present a summary of the anticoagulant and antithrombotic aptamers that have been identified and their stage of development and comment on the future of aptamer-based drug development to treat thrombosis. PMID:26882082

  10. Harnessing Aptamers to Overcome Challenges in Gluten Detection.

    Science.gov (United States)

    Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Lobo-Castañón, María Jesús

    2016-01-01

    Celiac disease is a lifelong autoimmune disorder triggered by foods containing gluten, the storage protein in wheat, rye, and barley. The rapidly escalating number of patients diagnosed with this disease poses a great challenge to both food industry and authorities to guarantee food safety for all. Therefore, intensive efforts are being made to establish minimal disease-eliciting doses of gluten and consequently to improve gluten-free labeling. These efforts depend to a high degree on the availability of methods capable of detecting the protein in food samples at levels as low as possible. Current analytical approaches rely on the use of antibodies as selective recognition elements. With limited sensitivity, these methods exhibit some deficiencies that compromise the accuracy of the obtained results. Aptamers provide an ideal alternative for designing biosensors for fast and selective measurement of gluten in foods. This article highlights the challenges in gluten detection, the current status of the use of aptamers for solving this problem, and what remains to be done to move these systems into commercial applications. PMID:27104578

  11. Isolation of peptide aptamers that inhibit intracellular processes

    OpenAIRE

    Blum, Jonathan H.; Dove, Simon L.; Hochschild, Ann; Mekalanos, John J.

    2000-01-01

    We have developed a method for isolation of random peptides that inhibit intracellular processes in bacteria. A library of random peptides expressed as fusions to Escherichia coli thioredoxin (aptamers) were expressed under the tight control of the arabinose-inducible PBAD promoter. A selection was applied to the library to isolate aptamers that interfered with the activity of thymidylate synthase (ThyA) in vivo. Expression of an aptamer isolated by this method resulted in a ThyA− phenotype t...

  12. Aptamers: Active Targeting Ligands for Cancer Diagnosis and Therapy

    OpenAIRE

    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...

  13. Clinical applications of nucleic acid aptamers in cancer

    OpenAIRE

    PEI, XIAOYU; Jun ZHANG; Liu, Jie

    2014-01-01

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

  14. Nucleic Acid Aptamers for Target Validation and Therapeutic Applications

    OpenAIRE

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

    2005-01-01

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

  15. Rapid Generation of Highly Specific Aptamers via Micromagnetic Selection

    OpenAIRE

    Qian, Jiangrong; Lou, Xinhui; Zhang, Yanting; Xiao, Yi; Soh, H. Tom

    2009-01-01

    Aptamers are nucleic acid-based reagents that bind to target molecules with high affinity and specificity. However, methods for generating aptamers from random combinatorial libraries (e.g., SELEX) are often labor-intensive and time-consuming. Recent studies suggest that microfluidic SELEX (M-SELEX) technology can accelerate aptamer isolation by enabling highly stringent selection conditions through the use of very small amounts of target molecules. We present here an alternative M-SELEX meth...

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

    OpenAIRE

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

    2011-01-01

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

  17. Aptamers targeting different functional groups of 17 beta-estradiol

    OpenAIRE

    Vanschoenbeek, Katrijn; Vanbrabant, Jeroen; Hosseinkhani, Baharak; Vermeeren, Veronique; Michiels, Luc

    2015-01-01

    Aptamers, short synthetic ssDNA or RNA molecules with a specific three-dimensional structure, are promising recognition elements in biosensor technology. In vitro generation of aptamers with high sensitivity and specificity toward a broad range of analytes has been achieved using the systematic evolution of ligands by exponential enrichment (SELEX) process. This iterative pathway of aptamer generation consists of sequential positive and counterselection steps. The present research aimed to se...

  18. 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.

  19. β-Conglutin dual aptamers binding distinct aptatopes.

    Science.gov (United States)

    Jauset Rubio, Miriam; Svobodová, Markéta; Mairal, Teresa; Schubert, Thomas; Künne, Stefan; Mayer, Günter; O'Sullivan, Ciara K

    2016-01-01

    An aptamer was previously selected against the anaphylactic allergen β-conglutin (β-CBA I), which was subsequently truncated to an 11-mer and the affinity improved by two orders of magnitude. The work reported here details the selection and characterisation of a second aptamer (β-CBA II) selected against a second aptatope on the β-conglutin target. The affinity of this second aptamer was similar to that of the 11-mer, and its affinity was confirmed by three different techniques at three independent laboratories. This β-CBA II aptamer in combination with the previously selected β-CBA I was then exploited to a dual-aptamer approach. The specific and simultaneous binding of the dual aptamer (β-CBA I and β-CBA II) to different sites of β-conglutin was confirmed using both microscale thermophoresis and surface plasmon resonance where β-CBA II serves as the primary capturing aptamer and β-CBA I or the truncated β-CBA I (11-mer) as the secondary signalling aptamer, which can be further exploited in enzyme-linked aptamer assays and aptasensors. PMID:26586159

  20. 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. PMID:27350620

  1. High Efficiency Acetylcholinesterase Immobilization on DNA Aptamer Modified Surfaces

    OpenAIRE

    Orada Chumphukam; Thao T. Le; Cass, Anthony E. G.

    2014-01-01

    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 ...

  2. Scintigraphic images of bacterial infection using aptamers directly labeled with {sup 99m}Tc

    Energy Technology Data Exchange (ETDEWEB)

    Santos, S.R.; Correa, C.R.; Andrade, A.S.R., E-mail: sararoberta7@hotmail.com, E-mail: crisrcorrea@gmail.com, E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil); Barros, A.L.B.; Diniz, S.O.F.; Cardoso, V.N., 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

    Staphylococcus aureus is specie of great medical importance and is the most commonly agent found in infections of soft tissues, bone infections and bone prostheses. In this study, aptamers selected to S. aureus were labeled by the direct method with {sup 99m}Tc and used for bacterial infection identification by scintigraphy. The radiolabeled aptamers radiochemical purity and stability were assessed by thin-layer chromatography (TLC). Three groups of Swiss mice (n=6) were used for the scintigraphic imaging studies. The first group was infected intramuscularly in the right thigh with S. aureus, the second group with C. albicans and the third group received zymosan to induce aseptic inflammation. After 24 h, radiolabeled aptamers (18 MBq) were injected by the tail vein. Scintigraphic images were acquired at 1 h and 4 h postinjection. The radiolabeling yield with {sup 99m}Tc was over 90%. The radiolabeled aptamers were stable in 0.9% saline, plasma and cysteine excess. The scintigraphic image profiles showed high uptake in the kidneys and bladder in all groups, indicating a main renal excretion consistent with the hydrophilic nature of the molecule. No accumulation of radioactivity was observed in the thyroid, stomach, liver and spleen, indicating acceptable levels of radiochemical impurities. The group infected with S. aureus showed a visible uptake in the infected right thigh at 1 h post-injection. For the control groups (C. albicans and zymosan) visible differences between the right and left thighs were not observed. The radiolabeled aptamers were able to distinguish aseptic inflammation from bacterial infection and bacterial from fungal infection. (author)

  3. Temporal Control of Aptamer Biosensors Using Covalent Self-Caging To Shift Equilibrium.

    Science.gov (United States)

    Tan, Zhesen; Feagin, Trevor A; Heemstra, Jennifer M

    2016-05-25

    Aptamer-based sensors provide a versatile and effective platform for the detection of chemical and biological targets. These sensors have been optimized to function in multiple formats, however, a remaining limitation is the inability to achieve temporal control over their sensing function. To overcome this challenge, we took inspiration from nature's ability to temporally control the activity of enzymes and protein receptors through covalent self-caging. We applied this strategy to structure-switching aptamer sensors through the installation of a cleavable linker between the two DNA fragments that comprise the sensor. Analogous to self-caged proteins, installation of this linker shifts the equilibrium of the aptamer sensor to disfavor target binding. However, activity can be restored in a time-resolved manner by cleavage of the linker. To demonstrate this principle, we chose a photocleavable linker and found that installation of the linker eliminates target binding, even at high target concentrations. However, upon irradiation with 365 nm light, sensor activity is restored with response kinetics that mirror those of the linker cleavage reaction. A key benefit of our approach is generality, which is demonstrated by grafting the photocleavable linker onto a different aptamer sensor and showing that an analogous level of temporal control can be achieved for sensing of the new target molecule. These results demonstrate that nature's self-caging approach can be effectively applied to non-natural receptors to provide precise temporal control over function. We envision that this will be of especially high utility for deploying aptamer sensors in biological environments. PMID:27159220

  4. Scintigraphic images of bacterial infection using aptamers directly labeled with 99mTc

    International Nuclear Information System (INIS)

    Staphylococcus aureus is specie of great medical importance and is the most commonly agent found in infections of soft tissues, bone infections and bone prostheses. In this study, aptamers selected to S. aureus were labeled by the direct method with 99mTc and used for bacterial infection identification by scintigraphy. The radiolabeled aptamers radiochemical purity and stability were assessed by thin-layer chromatography (TLC). Three groups of Swiss mice (n=6) were used for the scintigraphic imaging studies. The first group was infected intramuscularly in the right thigh with S. aureus, the second group with C. albicans and the third group received zymosan to induce aseptic inflammation. After 24 h, radiolabeled aptamers (18 MBq) were injected by the tail vein. Scintigraphic images were acquired at 1 h and 4 h postinjection. The radiolabeling yield with 99mTc was over 90%. The radiolabeled aptamers were stable in 0.9% saline, plasma and cysteine excess. The scintigraphic image profiles showed high uptake in the kidneys and bladder in all groups, indicating a main renal excretion consistent with the hydrophilic nature of the molecule. No accumulation of radioactivity was observed in the thyroid, stomach, liver and spleen, indicating acceptable levels of radiochemical impurities. The group infected with S. aureus showed a visible uptake in the infected right thigh at 1 h post-injection. For the control groups (C. albicans and zymosan) visible differences between the right and left thighs were not observed. The radiolabeled aptamers were able to distinguish aseptic inflammation from bacterial infection and bacterial from fungal infection. (author)

  5. Aptamer-Based K(+) Sensor: Process of Aptamer Transforming into G-Quadruplex.

    Science.gov (United States)

    Zhang, Dongju; Han, Juan; Li, Yunchao; Fan, Louzhen; Li, Xiaohong

    2016-07-14

    G-rich aptamers have been widely applied to develop various sensors for detecting proteins, small molecules, and cations, which is based on the target-induced conformational transfer from single strand to G-quadruplex. However, the transforming process is unclear. Here, with PW17 as an aptamer example, the forming process of G-quadruplex induced by K(+) is investigated by circular dichroism spectroscopy, electrospray ionization mass spectroscopy, and native gel electrophoresis. The results demonstrate that PW17 undergoes a conformational transforming process from loose and unstable to compact and stable G-quadruplex, which is strictly K(+) concentration-dependent. The process contains three stages: (1) K(+) (aptamer-based biosensers and logic devices. PMID:27322753

  6. Replacing antibodies with aptamers in lateral flow immunoassay.

    Science.gov (United States)

    Chen, Ailiang; Yang, Shuming

    2015-09-15

    Aptamers have been identified against various targets as a type of chemical or nucleic acid ligand by systematic evolution of ligands by exponential enrichment (SELEX) with high sensitivity and specificity. Aptamers show remarkable advantages over antibodies due to the nucleic acid nature and target-induced structure-switching properties and are widely used to design various fluorescent, electrochemical, or colorimetric biosensors. However, the practical applications of aptamer-based sensing and diagnostics are still lagging behind those of antibody-based tests. Lateral flow immunoassay (LFIA) represents a well established and appropriate technology among rapid assays because of its low cost and user-friendliness. The antibody-based platform is utilized to detect numerous targets, but it is always hampered by the antibody preparation time, antibody stability, and effect of modification on the antibody. Seeking alternatives to antibodies is an area of active research and is of tremendous importance. Aptamers are receiving increasing attention in lateral flow applications because of a number of important potential performance advantages. We speculate that aptamer-based LFIA may be one of the first platforms for commercial use of aptamer-based diagnosis. This review first gives an introduction to aptamer including the selection process SELEX with its focus on aptamer advantages over antibodies, and then depicts LFIA with its focus on aptamer opportunities in LFIA over antibodies. Furthermore, we summarize the recent advances in the development of aptamer-based lateral flow biosensing assays with the aim to provide a general guide for the design of aptamer-based lateral flow biosensing assays. PMID:25912679

  7. The Application of Aptamers for Immunohistochemistry.

    Science.gov (United States)

    Bauer, Michelle; Macdonald, Joanna; Henri, Justin; Duan, Wei; Shigdar, Sarah

    2016-06-01

    Immunohistochemistry has helped to make surgical pathology the "gold" standard for tumor diagnosis. However, given the numerous problems associated with the use of antibodies for the staining of cellular markers in paraffin-embedded tissues, there is a requirement for novel agents that have the advantages of antibodies, but with few of the disadvantages. Aptamers, which are chemical antibodies, are highly specific and sensitive, like their protein counterparts, but display few of the disadvantages. These molecules represent a unique reagent that has the potential to revolutionize the field of histopathological diagnostics. In this study, we present a review of some of the aptamers that have been validated for use in diagnoses and suggest some of the advantages to using these molecules in the future. PMID:26862683

  8. Prevention of Serpin Misfolding by RNA Aptamers.

    Science.gov (United States)

    Zhou, Xiaohua; Declerck, Paul J

    2016-06-23

    Owing to their structural flexibility, most serpins inhibit the cognate proteases in a fast and specific manner and also are susceptible to pathogenic misfolding. In this issue of Cell Chemical Biology, Madsen et al. (2016) report on the selection and characterization of an RNA aptamer that stabilizes α1-antichymotrypsin L55P mutant without interfering with the protease inhibitory activity. PMID:27341430

  9. Molecular-level Design of Heterogeneous Chiral Catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Gellman, Andrew John [Carnegie Mellon University; Sholl, David S. [Georgia Institute of Technology; Tysoe, Wilfred T. [University of Wisconsin - Milwaukee; Zaera, Francisco [University of California at Riverside

    2013-04-28

    Understanding and controlling selectivity is one of the key challenges in heterogeneous catalysis. Among problems in catalytic selectivity enantioselectivity is perhaps the most the most challenging. The primary goal of the project on “Molecular-level Design of Heterogeneous Chiral Catalysts” is to understand the origins of enantioselectivity on chiral heterogeneous surfaces and catalysts. The efforts of the project team include preparation of chiral surfaces, characterization of chiral surfaces, experimental detection of enantioselectivity on such surfaces and computational modeling of the interactions of chiral probe molecules with chiral surfaces. Over the course of the project period the team of PI’s has made some of the most detailed and insightful studies of enantioselective chemistry on chiral surfaces. This includes the measurement of fundamental interactions and reaction mechanisms of chiral molecules on chiral surfaces and leads all the way to rationale design and synthesis of chiral surfaces and materials for enantioselective surface chemistry. The PI’s have designed and prepared new materials for enantioselective adsorption and catalysis. Naturally Chiral Surfaces • Completion of a systematic study of the enantiospecific desorption kinetics of R-3-methylcyclohexanone (R-3-MCHO) on 9 achiral and 7 enantiomeric pairs of chiral Cu surfaces with orientations that span the stereographic triangle. • Discovery of super-enantioselective tartaric acid (TA) and aspartic acid (Asp) decomposition as a result of a surface explosion mechanism on Cu(643)R&S. Systematic study of super-enantiospecific TA and Asp decomposition on five enantiomeric pairs of chiral Cu surfaces. • Initial observation of the enantiospecific desorption of R- and S-propylene oxide (PO) from Cu(100) imprinted with {3,1,17} facets by L-lysine adsorption. Templated Chiral Surfaces • Initial observation of the enantiospecific desorption of R- and S-PO from Pt(111) and Pd(111

  10. Harnessing aptamers for electrochemical detection of endotoxin.

    Science.gov (United States)

    Kim, Sung-Eun; Su, Wenqiong; Cho, MiSuk; Lee, Youngkwan; Choe, Woo-Seok

    2012-05-01

    Lipopolysaccharide (LPS), also known as endotoxin, triggers a fatal septic shock; therefore, fast and accurate detection of LPS from a complex milieu is of primary importance. Several LPS affinity binders have been reported so far but few of them have proved their efficacy in developing electrochemical sensors capable of selectively detecting LPS from crude biological liquors. In this study, we identified 10 different single-stranded DNA aptamers showing specific affinity to LPS with dissociation constants (K(d)) in the nanomolar range using a NECEEM-based non-SELEX method. Based on the sequence and secondary structure analysis of the LPS binding aptamers, an aptamer exhibiting the highest affinity to LPS (i.e., B2) was selected to construct an impedance biosensor on a gold surface. The developed electrochemical aptasensor showed excellent sensitivity and specificity in the linear detection range from 0.01 to 1 ng/mL of LPS with significantly reduced detection time compared with the traditional Limulus amoebocyte lysate (LAL) assay. PMID:22370280

  11. Improving Molecular Level Chemical Speciation of Organic Aerosols

    Science.gov (United States)

    Worton, D. R.; Decker, M.; Isaacman, G. A.; Chan, A.; Wilson, K. R.; Goldstein, A. H.

    2013-12-01

    A substantial fraction of fine mode aerosols are organic with the majority formed in the atmosphere through oxidation of gas phase compounds emitted from a variety of natural and man-made sources. As a result, organic aerosols are comprised of thousands of individual organic species whose complexity increases exponentially with carbon number and degree of atmospheric oxidation. Chemical characterization of individual compounds present in this complex mixture provides information on sources and transformation processes that are critical for apportioning organic carbon from an often convoluted mixture of sources and to constrain oxidation mechanisms needed for atmospheric models. These compounds also affect the physical and optical properties of the aerosol but the vast majority remain unidentified and missing from published mass spectral libraries because of difficulties in separating and identifying them. We have developed improved methodologies for chemical identification in order to better understand complex environmental mixtures. Our approach has been to combine two-dimensional gas chromatography with high resolution time of flight mass spectrometry (GC×GC-HRTOFMS) and both traditional electron ionization (EI) and vacuum ultraviolet (VUV) photoionization. GC×GC provides improved separation of individual compounds over traditional one dimensional GC and minimizes co-elution of peaks resulting in mass spectra that are virtually free of interferences. VUV ionization is a ';soft' ionization technique that reduces fragmentation and enhances the abundance of the parent or molecular ion, which when combined with high resolution mass spectrometry can provide molecular formulas for chromatographic peaks. We demonstrate our methodology by applying it to identify more than 500 individual compounds in aerosol filter samples collected at Blodgett Forest, a rural site in the Sierra Nevada Mountains. Using the EI NIST mass spectral library and molecular formulas determined

  12. Rapid one-step selection method for generating nucleic acid aptamers: development of a DNA aptamer against α-bungarotoxin.

    Directory of Open Access Journals (Sweden)

    Lasse H Lauridsen

    Full Text Available 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®, an inhibitor of vascular endothelial growth factor (VEGF for the treatment of age related macular degeneration (AMD. Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period. PRINCIPAL FINDINGS: Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM. 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 of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.

  13. A Complex Approach for Unravelling Musaceae Phylogeny at Molecular Level

    Czech Academy of Sciences Publication Activity Database

    Němcová, Pavla; Hřibová, Eva; Valárik, Miroslav; Doležel, Jaroslav

    2011-01-01

    Roč. 897, SEP 14 (2011), s. 139-142. ISSN 0567-7572. [INTERNATIONAL ISHS-PROMUSA SYMPOSIUM ON GLOBAL PERSPECTIVES ON ASIAN CHALLENGES. Guangzhou, 14.08.2009-16.08.2009] R&D Projects: GA AV ČR IAA600380703 Institutional support: RVO:61389030 Keywords : DArT * low-copy genes * molecular phylogenetics Subject RIV: EF - Botanics http://www.actahort.org/books/897/897_14.htm

  14. Biotin Sensing at the Molecular Level1–3

    OpenAIRE

    Beckett, Dorothy

    2009-01-01

    Biotin influences transcription in organisms from bacteria to humans. The enzyme, biotin protein ligase, which catalyzes post-transcriptional biotin addition to biotin-dependent carboxylases, plays a central roll in transmitting the demand for biotin to gene expression. The molecular mechanism of this communication in bacteria is well understood and involves competing protein:protein interactions. Biochemical measurements indicate that this competition is kinetically controlled. In humans, th...

  15. 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.

  16. Chemical maturation of a bivalent aptamer by single domain variation

    DEFF Research Database (Denmark)

    Rohrbach, Falk; Fatthalla, Maha I; Kupper, Tina;

    2012-01-01

    Two-pronged attack: We describe the maturation of a bivalent aptamer by a chemically driven two-step process. From an improved monovalent aptamer subdomain that had been modified by polycyclic aromatic hydrocarbons at individual positions, a mature bivalent variant with superior activities to its...

  17. 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.

  18. A fluorescent sandwich assay for thrombin using aptamer modified magnetic beads and quantum dots

    International Nuclear Information System (INIS)

    We describe an aptamer-based sandwich assay for thrombin by using a pair of thrombin-binding aptamers, namely one 15-mer aptamer (denoted as Apt15) and one 29-mer aptamer (denoted as Apt29). Either Apt29 or Apt15 can be used as capture aptamers on magnetic beads or reporter aptamers on the quantum dots to form the sandwich complex. Detection of thrombin is achieved by the fluorescent measurement of quantum dots in the sandwich complex. The choice of capture aptamers and reporter aptamers, and the effect of the addition order of the aptamers modified magnetic beads and the aptamers modified quantum dots were investigated. Detection of 0.05 nM thrombin was accomplished. The proteins hemoglobin, lysozyme, and transferrin did not interfere in this assay. (author)

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

    Science.gov (United States)

    Kusser, W

    2000-03-01

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

  20. Optical nanosensor architecture for cell-signaling molecules using DNA aptamer-coated carbon nanotubes.

    Science.gov (United States)

    Cha, Tae-Gon; Baker, Benjamin A; Sauffer, M Dane; Salgado, Janette; Jaroch, David; Rickus, Jenna L; Porterfield, D Marshall; Choi, Jong Hyun

    2011-05-24

    We report a novel optical biosensor platform using near-infrared fluorescent single-walled carbon nanotubes (SWNTs) functionalized with target-recognizing aptamer DNA for noninvasively detecting cell-signaling molecules in real time. Photoluminescence (PL) emission of aptamer-coated SWNTs is modulated upon selectively binding to target molecules, which is exploited to detect insulin using an insulin-binding aptamer (IBA) as a molecular recognition element. We find that nanotube PL quenches upon insulin recognition via a photoinduced charge transfer mechanism with a quenching rate of k(q) = 5.85 × 10(14) M(-1) s(-1) and a diffusion-reaction rate of k(r) = 0.129 s(-1). Circular dichroism spectra reveal for the first time that IBA strands retain a four-stranded, parallel guanine quadruplex conformation on the nanotubes, ensuring target selectivity. We demonstrate that these IBA-functionalized SWNT sensors incorporated in a collagen extracellular matrix (ECM) can be regenerated by removing bound analytes through enzymatic proteolysis. As proof-of-concept, we show that the SWNT sensors embedded in the ECM promptly detect insulin secreted by cultured pancreatic INS-1 cells stimulated by glucose influx and report a gradient contour of insulin secretion profile. This novel design enables new types of label-free assays and noninvasive, in situ, real-time detection schemes for cell-signaling molecules. PMID:21520951

  1. Gold-Coated Superparamagnetic Nanoparticles for Single Methyl Discrimination in DNA Aptamers

    Directory of Open Access Journals (Sweden)

    Maria Tintoré

    2015-11-01

    Full Text Available Au- and iron-based magnetic nanoparticles (NPs are promising NPs for biomedical applications due to their unique properties. The combination of a gold coating over a magnetic core puts together the benefits from adding the magnetic properties to the robust chemistry provided by the thiol functionalization of gold. Here, the use of Au-coated magnetic NPs for molecular detection of a single methylation in DNA aptamer is described. Binding of α-thrombin to two aptamers conjugated to these NPs causes aggregation, a phenomenon that can be observed by UV, DLS and MRI. These techniques discriminate a single methylation in one of the aptamers, preventing aggregation due to the inability of α-thrombin to recognize it. A parallel study with gold and ferromagnetic NPs is detailed, concluding that the Au coating of FexOy NP does not affect their performance and that they are suitable as complex biosensors. These results prove the high detection potency of Au-coated SPIONs for biomedical applications especially for DNA repair detection.

  2. Gold-Coated Superparamagnetic Nanoparticles for Single Methyl Discrimination in DNA Aptamers

    Science.gov (United States)

    Tintoré, Maria; Mazzini, Stefania; Polito, Laura; Marelli, Marcello; Latorre, Alfonso; Somoza, Álvaro; Aviñó, Anna; Fàbrega, Carme; Eritja, Ramon

    2015-01-01

    Au- and iron-based magnetic nanoparticles (NPs) are promising NPs for biomedical applications due to their unique properties. The combination of a gold coating over a magnetic core puts together the benefits from adding the magnetic properties to the robust chemistry provided by the thiol functionalization of gold. Here, the use of Au-coated magnetic NPs for molecular detection of a single methylation in DNA aptamer is described. Binding of α-thrombin to two aptamers conjugated to these NPs causes aggregation, a phenomenon that can be observed by UV, DLS and MRI. These techniques discriminate a single methylation in one of the aptamers, preventing aggregation due to the inability of α-thrombin to recognize it. A parallel study with gold and ferromagnetic NPs is detailed, concluding that the Au coating of FexOy NP does not affect their performance and that they are suitable as complex biosensors. These results prove the high detection potency of Au-coated SPIONs for biomedical applications especially for DNA repair detection. PMID:26593913

  3. Aptamers and Their Significant Role in Cancer Therapy and Diagnosis

    Directory of Open Access Journals (Sweden)

    Joy Sebastian Prakash

    2015-09-01

    Full Text Available Aptamers are nucleic acid/peptide molecules that can be generated by a sophisticated, well-established technique known as Systematic Evolution of Ligands by EXponential enrichment (SELEX. Aptamers can interact with their targets through structural recognition, as in antibodies, though with higher specificity. With this added advantage, they can be made useful for clinical applications such as targeted therapy and diagnosis. In this review, we have discussed the steps involved in SELEX process and modifications executed to attain high affinity nucleic acid aptamers. Moreover, our review also highlights the therapeutic applications of aptamer functionalized nanoparticles and nucleic acids as chemo-therapeutic agents. In addition, we have described the development of “aptasensor” in clinical diagnostic application for detecting cancer cells and the use of aptamers in different routine imaging techniques, such as Positron Emission Tomography/Computed Tomography, Ultrasound, and Magnetic Resonance Imaging.

  4. Fit for the Eye: Aptamers in Ocular Disorders

    Science.gov (United States)

    Drolet, Daniel W.; Green, Louis S.; Gold, Larry

    2016-01-01

    For any new class of therapeutics, there are certain types of indications that represent a natural fit. For nucleic acid ligands in general, and aptamers in particular, the eye has historically been an attractive site for therapeutic intervention. In this review, we recount the discovery and early development of three aptamers designated for use in ophthalmology, one approved (Macugen), and two in late-stage development (Fovista and Zimura). Every one of these molecules was originally intended for other indications. Key improvements in technology, specifically with regard to libraries used for in vitro selection and subsequent chemical optimization of aptamers, have played an important role in allowing the identification of development candidates with suitable properties. The lessons learned from the selection of these molecules are valuable for informing us about the many remaining opportunities for aptamer-based therapeutics in ophthalmology as well as for identifying additional indications for which aptamers as a class of therapeutics have distinct advantages. PMID:26757406

  5. 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.

  6. Fit for the Eye: Aptamers in Ocular Disorders.

    Science.gov (United States)

    Drolet, Daniel W; Green, Louis S; Gold, Larry; Janjic, Nebojsa

    2016-06-01

    For any new class of therapeutics, there are certain types of indications that represent a natural fit. For nucleic acid ligands in general, and aptamers in particular, the eye has historically been an attractive site for therapeutic intervention. In this review, we recount the discovery and early development of three aptamers designated for use in ophthalmology, one approved (Macugen), and two in late-stage development (Fovista and Zimura). Every one of these molecules was originally intended for other indications. Key improvements in technology, specifically with regard to libraries used for in vitro selection and subsequent chemical optimization of aptamers, have played an important role in allowing the identification of development candidates with suitable properties. The lessons learned from the selection of these molecules are valuable for informing us about the many remaining opportunities for aptamer-based therapeutics in ophthalmology as well as for identifying additional indications for which aptamers as a class of therapeutics have distinct advantages. PMID:26757406

  7. Particle Display: A Quantitative Screening Method for Generating High-Affinity Aptamers**

    OpenAIRE

    Wang, J.; Gong, Q; N Maheshwari; Eisenstein, M; Arcila, ML; Kosik, KS; Soh, HT

    2014-01-01

    We report an aptamer discovery technology that reproducibly yields higher affinity aptamers in fewer rounds compared to conventional selection. Our method (termed particle display) transforms libraries of solution-phase aptamers into "aptamer particles", each displaying many copies of a single sequence on its surface. We then use fluorescence-activated cell sorting (FACS) to individually measure the relative affinities of >108 aptamer particles and sort them in a high-throughput manner. Throu...

  8. 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)

  9. 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) for...... the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection......, possibly in one-step, technique is required for developing aptamers in limited time period. Principal Findings: Herein, we present a simple one-step selection of DNA aptamers against alpha-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing...

  10. Design strategies for the molecular level synthesis of supported catalysts.

    Science.gov (United States)

    Wegener, Staci L; Marks, Tobin J; Stair, Peter C

    2012-02-21

    Supported catalysts, metal or oxide catalytic centers constructed on an underlying solid phase, are making an increasingly important contribution to heterogeneous catalysis. For example, in industry, supported catalysts are employed in selective oxidation, selective reduction, and polymerization reactions. Supported structures increase the thermal stability, dispersion, and surface area of the catalyst relative to the neat catalytic material. However, structural and mechanistic characterization of these catalysts presents a formidable challenge because traditional preparations typically afford complex mixtures of structures whose individual components cannot be isolated. As a result, the characterization of supported catalysts requires a combination of advanced spectroscopies for their characterization, unlike homogeneous catalysts, which have relatively uniform structures and can often be characterized using standard methods. Moreover, these advanced spectroscopic techniques only provide ensemble averages and therefore do not isolate the catalytic function of individual components within the mixture. New synthetic approaches are required to more controllably tailor supported catalyst structures. In this Account, we review advances in supported catalyst synthesis and characterization developed in our laboratories at Northwestern University. We first present an overview of traditional synthetic methods with a focus on supported vanadium oxide catalysts. We next describe approaches for the design and synthesis of supported polymerization and hydrogenation catalysts, using anchoring techniques which provide molecular catalyst structures with exceptional activity and high percentages of catalytically significant sites. We then highlight similar approaches for preparing supported metal oxide catalysts using atomic layer deposition and organometallic grafting. Throughout this Account, we describe the use of incisive spectroscopic techniques, including high

  11. Aptamer-Based Analysis: A Promising Alternative for Food Safety Control

    Directory of Open Access Journals (Sweden)

    Sonia Amaya-González

    2013-11-01

    Full Text Available Ensuring food safety is nowadays a top priority of authorities and professional players in the food supply chain. One of the key challenges to determine the safety of food and guarantee a high level of consumer protection is the availability of fast, sensitive and reliable analytical methods to identify specific hazards associated to food before they become a health problem. The limitations of existing methods have encouraged the development of new technologies, among them biosensors. Success in biosensor design depends largely on the development of novel receptors with enhanced affinity to the target, while being stable and economical. Aptamers fulfill these characteristics, and thus have surfaced as promising alternatives to natural receptors. This Review describes analytical strategies developed so far using aptamers for the control of pathogens, allergens, adulterants, toxins and other forbidden contaminants to ensure food safety. The main progresses to date are presented, highlighting potential prospects for the future.

  12. Evaluation of aptamers labelled with 99mTc for identification of Staphylococcus aureus bacteria

    International Nuclear Information System (INIS)

    Staphylococcus aureus is specie of great medical importance because it is often associated with many infections in humans. This bacterium can cause diseases ranging from simple infections to life-threatening infections such as endocarditis, pneumonia, meningitis, toxic shock syndrome, septicemia, osteomyelitis, among others. S. aureus is the most commonly agent found in infections of the skin and soft tissues, bone infections and bone prostheses. The difficulty in early detection of specific foci caused by bacteria has raised the need to search for new techniques for this purpose. Diagnosis by scintigraphy has advantages over other methods because it is able to identify damage tissues without the need of invasive procedures and is able to perform an early diagnosis even before anatomic changes. Thus, nuclear medicine could contribute to an accurate diagnosis of bacterial infections, since specific radiopharmaceuticals were developed. Aptamers are oligonucleotides that have high affinity and specificity for their molecular targets and are emerging as a new class of molecules for radiopharmaceuticals development. Radiolabeled aptamers specific to the infectious agents, could give a significant contribution to the infection diagnosis by scintigraphy. In this study, aptamers selected to S. aureus were labeled with 99mTc and used for the bacteria identification in vitro and in vivo. The aptamers labeled with 32P and incubated in vitro with S. aureus cells showed high affinity for the bacterial cells when compared with the library of oligonucleotides with random sequences used as control. The aptamers labeled with 99mTc also showed affinity for S. aureus cells when compared with the library, but unspecific binding was also verified. The 99mTc labelled aptamers were stable in 0.9% saline, plasma of Swiss mice and in excess of cysteine. The in vivo biodistribution studies using Swiss mice with intramuscular infection in the right thigh showed that the aptamers labeled with

  13. 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.

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

    International Nuclear Information System (INIS)

    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

  15. Molecular-Level Organization of the Tear Film Lipid Layer: A Molecular Dynamics Simulation Study

    Czech Academy of Sciences Publication Activity Database

    Wizert, A.; Iskander, D. R.; Jungwirth, Pavel; Cwiklik, Lukasz

    Elsevier. Roč. 106, č. 2 (2014), 710A. ISSN 0006-3495. [Annual Meeting of the Biophysical Society /58./. 15.02.2014-19.02.2014, San Francisco] Institutional support: RVO:61388963 ; RVO:61388955 Keywords : tear film * lipid layer * molecular dynamics simulations Subject RIV: BO - Biophysics

  16. Renormalized molecular levels in a Sc3N@C-80 molecular electronic device

    DEFF Research Database (Denmark)

    Larade, Brian; Taylor, Jeremy Philip; Zheng, Q. R.;

    2001-01-01

    We address several general questions about quantum transport through molecular systems by an ab initio analysis of a scandium-nitrogen doped C-80 metallofullerene device. Charge transfer from the Sc3N is found to drastically change the current-voltage characteristics: the current through the Sc3N...

  17. Radiation damage at the molecular level: Nanodosimetry; Dano por radiacion a nivel molecular: nanodosimetria

    Energy Technology Data Exchange (ETDEWEB)

    Blanco, F.; Munoz, A.; Lagares, J. I.; Nunez, L.; Garcia, G.

    2013-07-01

    One of the main practical use of the model is its use as a tool of nanodosimetry which basically consists in characterizing the effect of radiation on nano volumes (comparable to the DNA of volumes) in terms of link breaks and molecular dissociations. (Author)

  18. Molecular and cellular level of action of digitalis.

    Science.gov (United States)

    Charlemagne, D

    1993-04-01

    The pharmacological receptor of cardiac glycosides is the Na+/K(+)-ATPase which consists of a catalytic alpha (M(r) = 112,000) and glycosylated beta (M(r) = 35,000) subunit. The enzyme is responsible for the vectorial transport across the sarcolemma of three Na+ ions outward and two K+ ions inward against their electrochemical gradient. Specific inhibition of the Na+ pump by digitalis induces a positive inotropic effect by increasing the intracellular Na+ concentration which in turn induces an increase in the intracellular Ca2+ concentration by the Na+/Ca2+ exchange and an increase in the Ca2+ pool of the sarcoplasmic reticulum; toxic effects are observed at higher doses of cardiac glycosides leading to spontaneous calcium release from the sarcoplasmic reticulum. Three isoforms of the alpha catalytic subunit have been identified by molecular cloning. They share a high homology in the deduced amino acid sequence with eight transmembrane domains. The ouabain binding domain is located on the extracellular side and ouabain sensitivity depends mainly on the two residues at the border of the first extracellular domain. The isoforms differed by their ouabain sensitivity, are expressed in a tissue-specific and hormonally-regulated manner. Moreover, expression of the isoforms and their ouabain sensitivity vary from species to species with an alpha 1 isoform of very low affinity being the major isoform (80%) in the adult rat heart and an alpha 1 isoform of high affinity representing 50% of total alpha mRNA abundance in the human heart. Therefore the effect of digitalis on the heart depends mainly on the isoform which is expressed and on the regulation of their expression according to age, hormonal influence and pathology. PMID:7684015

  19. The challenges for molecular nutrition research 4: the "nutritional systems biology level"

    NARCIS (Netherlands)

    Ommen, B. van; Cavallieri, D.; Roche, H.M.; Klein, U.I.; Daniel, H.

    2008-01-01

    Nutritional systems biology may be defined as the ultimate goal of molecular nutrition research, where all relevant aspects of regulation of metabolism in health and disease states at all levels of its complexity are taken into account to describe the molecular physiology of nutritional processes. T

  20. Molecular-Level Design of Heterogeneous Chiral Catalysis

    International Nuclear Information System (INIS)

    , and the development of ways to imprint chiral centers on achiral solid surfaces. Chiral catalysis is not only a problem of great importance in its own right, but also the ultimate test of how to control selectivity in catalysis. The time is ripe for fundamental work in heterogeneous chiral catalysis to provide the U.S. with a leadership role in developing the next generation of catalytic processes for medicinal and agrochemical manufacturing. Our team provides the required expertise for a synergistic and comprehensive integration of physical and chemical experimentation with solid state and molecular reactivity theories to solve this problem.

  1. Molecular-Level Design of Heterogeneous Chiral Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Francisco Zaera

    2012-03-21

    , and the development of ways to imprint chiral centers on achiral solid surfaces. Chiral catalysis is not only a problem of great importance in its own right, but also the ultimate test of how to control selectivity in catalysis. The time is ripe for fundamental work in heterogeneous chiral catalysis to provide the U.S. with a leadership role in developing the next generation of catalytic processes for medicinal and agrochemical manufacturing. Our team provides the required expertise for a synergistic and comprehensive integration of physical and chemical experimentation with solid state and molecular reactivity theories to solve this problem.

  2. Fine-tuning molecular energy levels by nonresonant laser pulses

    CERN Document Server

    Lemeshko, Mikhail

    2010-01-01

    We evaluate the shifts imparted to vibrational and rotational levels of a linear molecule by a nonresonant laser field at intensities of up to 10^12 W/cm^2. Both types of shift are found to be either positive or negative, depending on the initial rotational state acted upon by the field. An adiabatic field-molecule interaction imparts a rotational energy shift which is negative and exceeds the concomitant positive vibrational shift by a few orders of magnitude. The rovibrational states are thus pushed downward in such a field. A nonresonant pulsed laser field that interacts nonadiabatically with the molecule is found to impart rotational and vibrational shifts of the same order of magnitude. The nonadiabatic energy transfer occurs most readily at a pulse duration which amounts to about a tenth of the molecule's rotational period, and vanishes when the sudden regime is attained for shorter pulses. We applied our treatment to the much studied 87Rb_2 molecule in the last bound vibrational levels of its lowest si...

  3. Cellular delivery of shRNA using aptamer-conjugated PLL-alkyl-PEI nanoparticles.

    Science.gov (United States)

    Askarian, Saeedeh; Abnous, Khalil; Taghavi, Sahar; Oskuee, Reza Kazemi; Ramezani, Mohammad

    2015-12-01

    Introduction of an efficient gene delivery vector is still the main challenge of gene therapy. Both polyethylenimine (PEI) and poly(l-lysine) (PLL) comprise disadvantages which limited their application. To explore whether their deficiencies could be compensated by preparing copolymers consisting of both PLL and PEI, we generated several combinations of PLL-alkyl-PEI copolymers conjugated to aptamer and evaluated their both gene delivery efficiency and down-regulation of Bcl-XL, an anti-apoptotic gene, in lung cancer cell line. PLL was conjugated to either 10% or 50% of PEI by grafting different percentages of PEI to alkylated-PLL as core. The properties of modified polymers including size, surface charge density, DNA condensation ability, buffering capacity and cytotoxicity were evaluated. According to transfection results, aptamer conjugated PLL-alkyl-10%-PEI (PLPE8%) was selected for further gene silencing study by plasmid shRNA. Decrease in Bcl-XL gene expression was estimated by both RT-PCR and western-blot experiments. The obtained results revealed that the new copolymers had appropriate nano-scale size (117-128 nm) even after aptamer conjugation (168-183 nm). Moreover, they exhibited increased transfection efficiencies by up to 1.8-5 folds and acceptable cytotoxicity. The apoptosis was induced in transfected cells by shRNA-aptamer-copolymer due to the down-regulation of mRNA and protein levels. This study suggested a new vector for targeted non-viral gene delivery with high transfection efficiency in lung cancer or pulmonary systems. PMID:26433348

  4. Aptamers: A Promising Tool for Ochratoxin A Detection in Food Analysis

    Directory of Open Access Journals (Sweden)

    Akhtar Hayat

    2013-11-01

    Full Text Available The contamination of food and feed by mycotoxins has become an increasingly serious problem. Mycotoxins represent a major risk to human and animal health, as well as economics. Herein, we focus on Ochratoxin A (OTA, which is one of the most common mycotoxins contaminating feed and foodstuffs. OTA is a secondary metabolite produced by various Aspergillus and Penicillium strains. Upon ingestion, OTA has a number of acute and chronic toxic effects. It is nephrotoxic, teratogenic, immunosuppressive, and carcinogenic (group 2B. As a consequence, some regulatory limits have been introduced on the levels of OTA in several commodities. The toxic nature of OTA demands highly sensitive and selective monitoring techniques to protect human and animal health. As alternative to traditional analytical techniques, biochemical methods for OTA analysis have attained great interest in the last few decades. They are mainly based on the integration of antibodies or aptamers as biorecognition elements in sensing platforms. However, aptamers have gained more attention in affinity-based assays because of their high affinity, specificity, stability, and their easy chemical synthesis. In this brief review, we present an overview of aptamer-based assays and their applications in OTA purification and detection, appeared in the literature in the last five years.

  5. Synthesis of heterocycles: Indolo (2,1-a) isoquinolines, renewables, and aptamer ligands for cellular imaging

    Energy Technology Data Exchange (ETDEWEB)

    Beasley, Jonathan [Ames Laboratory (AMES), Ames, IA (United States)

    2013-01-01

    In this thesis, we explore both total syntheses and methodologies of several aromatic heterocyclic molecules. Extensions of the Kraus indole synthesis toward 2-substituted and 2,3-disubstituted indoles, as well as biologically attractive indolo[2,1-a]isoquinolines are described. Recent renewable efforts directed to commodity maleic acid and the first reported furan-based ionic liquids are described. Our total synthesis of mRNA aptamer ligand PDC-Gly, and its dye coupled forms, plus aminoglycoside dye coupled ligands used in molecular imaging, are described.

  6. An aptamer based lateral flow strip for on-site rapid detection of ochratoxin A in Astragalus membranaceus.

    Science.gov (United States)

    Zhou, Weilu; Kong, Weijun; Dou, Xiaowen; Zhao, Ming; Ouyang, Zhen; Yang, Meihua

    2016-06-01

    An aptamer based lateral flow strip based on competitive format was developed for on-site rapid detection of ochratoxin A (OTA) in Astragalus membranaceus. Some crucial parameters that might influence the sensitive detection, such as the characterization of the colloidal gold, size and shape of gold nanoparticles (AuNPs), amount of AuNPs-aptamer conjugate, migration rate and the addition amount of methanol, were investigated to provide the optimum assay performance. To perform the test, 1g sample was extracted with 2.5mL of methanol-water (80:20, v/v) and diluted by 4-fold running buffer to eliminate the matrix and methanol interferences. Under optimized conditions, the aptamer-based assay showed a visual limit of detection (LOD) of 1ngmL(-1), and with no significant cross-reactivity with several homologous toxins. The whole detection could be completed within 15min without special equipment because of available visual results. One out of nine A. membranaceus samples was found to be positive of OTA, which was in a good agreement with those obtained from LC-MS/MS analysis. The results demonstrated that the aptamer-based lateral flow assay could be used as a rapid, reliable, cost-effective and robust on-site screening technique for mycotoxins at trace level in complex matrices without special instrumentation. PMID:27085019

  7. Aptamer biosensor for dopamine based on a gold electrode modified with carbon nanoparticles and thionine labeled gold nanoparticles as probe

    International Nuclear Information System (INIS)

    We describe a biosensor for dopamine that is based on the use of a gold electrode modified with carbon nanoparticles (CNPs) coupled to thionine labeled gold nanoparticles (AuNPs) acting as signal amplifiers. The biosensor was constructed by first modifying the CNPs on the gold electrode and adsorbing the thionine on the surface of the AuNPs, and then linking the complementary strand of the dopamine aptamer to the AuNPs via gold-thiol chemistry. Next, dopamine aptamer is added and the duplex is formed on the surface. On addition of a sample containing dopamine, it will interact with aptamer and cause the release of the electrochemical probe which then will be adsorbed on the surface of the CNP-modified gold electrode and detected by differential pulse voltammetry. The current is linearly related to the concentration of dopamine in the 30 nM to 6.0 μM ranges. The detection limit is as low as 10 nM, and the RSD is 3.1 % at a 0.3 μM level (for n = 11). The protocol was successfully applied to the determination of dopamine in spiked human urine samples. We perceive that this method holds promise as a widely applicable platform for aptamer-based electrochemical detection of small molecules. (author)

  8. Determination of the invA gene of Salmonella using surface plasmon resonance along with streptavidin aptamer amplification

    International Nuclear Information System (INIS)

    We have developed a sensitive method for the determination of Salmonella by integrating a streptavidinylated aptamer (SA-aptamer) as a signal amplification unit along with a modified asymmetric polymerase chain reaction (PCR) technique into the surface of an SPR sensor chip. The gold film of the sensor was first modified with a thiolated probe, and the target sequence and SA-aptamer were then induced to form a sandwich structure. If SA is added, the SA-aptamer forms a complex with SA which will amplify the signal. Under optimal conditions, this sensing scheme has a linear response in the 50 pM to 200 nM range, and the lower detection limit is 20 pM (for a synthetic target sequence). This strategy was successfully applied to the determination of Salmonella bacteria at levels as low as 60 CFU mL−1. This biosensor is sensitive, selective and highly stable. These features make this strategy a promising and powerful screening tool to detect pathogens in food, and in clinical and environmental samples. (author)

  9. 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.

  10. The eleven-year switch of peptide aptamers

    OpenAIRE

    Colas, Pierre

    2008-01-01

    Peptide aptamers are combinatorial recognition proteins that were introduced more than ten years ago. They have since found many applications in fundamental and therapeutic research, including their recent use in microarrays to detect individual proteins from complex mixtures.

  11. Aptamer based electrochemical sensors for emerging environmental pollutants

    Directory of Open Access Journals (Sweden)

    Akhtar HAYAT

    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.

  12. X-Aptamers: A bead-based selection method for random incorporation of drug-like moieties onto next-generation aptamers for enhanced binding

    OpenAIRE

    He, WeiGuo; Elizondo-Riojas, Miguel-Angel; LI, XIN; Lokesh, Ganesh Lakshmana Rao; Somasunderam, Anoma; Thiviyanathan, Varatharasa; Volk, David E.; Durland, Ross H.; Englehardt, Johnnie; Cavasotto, Claudio N.; Gorenstein, David G.

    2012-01-01

    By combining pseudo-random bead-based aptamer libraries with conjugation chemistry, we have created next-generation aptamers, X-aptamers (XAs). Several X ligands can be added in a directed or random fashion to the aptamers to further enhance their binding affinities to the target proteins. Here we describe the addition of a drug (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid) demonstrated to bind to CD44-HABD, to a complete monothioate backbone substituted aptamer to increase its binding affini...

  13. Thrombin Binding Aptamer, More than a Simple Aptamer: Chemically Modified Derivatives and Biomedical Applications

    OpenAIRE

    Aviñó, Anna Maria; Eritja Casadellà, Ramón; Fàbrega, Carme; Tintoré, María

    2012-01-01

    The thrombin binding aptamer (TBA) is a well characterized chair-like, antiparallel quadruplex structure that binds specifically to thrombin at nanomolar concentrations and therefore it has interesting anticoagulant properties. In this article we review the research involved in the development of new TBA derivatives with improved anticoagulant properties as well as the use of the TBA as a model compound for the study of quadruplex structures. Specifically, we describe the impact of modified n...

  14. Aptamers in Diagnostics and Treatment of Viral Infections

    OpenAIRE

    Tomasz Wandtke; Joanna Woźniak; Piotr Kopiński

    2015-01-01

    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 o...

  15. Identification of Epithelial Ovarian Tumor-Specific Aptamers

    OpenAIRE

    Benedetto, Gregory; Hamp, Timothy J; Wesselman, Peter J.; Richardson, Christine

    2015-01-01

    Ovarian cancer is often diagnosed in late stages with few treatment options and poor long-term prognosis. New clinical tools for early detection of ovarian malignancies will significantly help reduce mortality and improve current long-term survival rates. The objective of this work was to identify ovarian tumor-specific single-stranded DNA aptamers that bind to malignant ovarian tumor cells and internalize with high affinity and specificity. Aptamers can identify unique tumor biomarkers, can ...

  16. P47THE IDENTIFICATION OF NOVEL APTAMERS TO GLIOMA

    OpenAIRE

    Norris, Karl; Shaw, Lisa; Alder, Jane Elizabeth; Lawrence, Clare Louise

    2014-01-01

    INTRODUCTION: Glioma represent less than 2% of all cancer cases in the UK, however, 80% of these tumours are glioblastoma (GBM). GBM is a highly aggressive tumour with poor patient survival rates, despite treatment involving surgery and radiotherapy with adjuvant chemotherapy. Delivery systems that have the ability to distinguish neoplasms from non-cancerous tissues, such as aptamers, may improve patient outcome. Aptamers have previously been shown to selectively target glioma cell lines. Apt...

  17. Aptamers Binding to c-Met Inhibiting Tumor Cell Migration.

    Directory of Open Access Journals (Sweden)

    Birgit Piater

    Full Text Available The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF. Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2'-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX. CLN64 and a previously described single-stranded DNA (ssDNA aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding.

  18. 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.

  19. 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.

  20. Aptamer carbon nanodot sandwich used for fluorescent detection of protein.

    Science.gov (United States)

    Xu, Bailu; Zhao, Chuanqi; Wei, Weili; Ren, Jinsong; Miyoshi, Daisuke; Sugimoto, Naoki; Qu, Xiaogang

    2012-12-01

    Carbon nanodots (C-Dots) have attracted growing interest in recent years due to their low cost, ready scalability, excellent chemical stability, biocompatibility, colloidal stability, and resilience of photoluminescence. They have been employed as novel, ideal fluorescent probes for bio-imaging and smart sensing. In addition, taking advantage of their low-cytotoxicity, C-Dots have potential applications in biochemical and cell biological fields. Herein, we present the first assay with aptamer-functionalized C-Dots as a sensory platform for protein detection. The presence of thrombin can induce the aptamer-modified fluorescent C-Dots to form a sandwich structure with aptamer-functionalized silica nanoparticles through specific protein/aptamer interaction. The assay shows high specificity toward thrombin. A detection limit of 1 nM is obtained, which is significantly improved as compared to that of many previously reported fluorescence-based thrombin detection assays. Using other modified aptamers and antibodies instead of thrombin binding aptamers, this strategy may offer a suitable approach for detection of other proteins in biological, pharmaceutical and nano-mechanical applications. PMID:23050264

  1. Aptamer-based SERRS sensor for thrombin detection.

    Science.gov (United States)

    Cho, Hansang; Baker, Brian R; Wachsmann-Hogiu, Sebastian; Pagba, Cynthia V; Laurence, Ted A; Lane, Stephen M; Lee, Luke P; Tok, Jeffrey B H

    2008-12-01

    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 alpha-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'-capped, 3'-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. PMID:19367849

  2. Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas.

    Directory of Open Access Journals (Sweden)

    David Kryza

    Full Text Available The human Matrix MetalloProtease-9 (hMMP-9 is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B, fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of 99mTc-MAG-aptamer and 111In-DOTA-F3B. 99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of 111In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05 higher for 111In-DOTA-F3B with 2.0%ID/g than for the 111In-DOTA-control oligonucleotide (0.7%ID/g with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor.

  3. Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas

    Science.gov (United States)

    Kryza, David; Debordeaux, Frédéric; Azéma, Laurent; Hassan, Aref; Paurelle, Olivier; Schulz, Jürgen; Savona-Baron, Catherine; Charignon, Elsa; Bonazza, Pauline; Taleb, Jacqueline; Fernandez, Philippe; Janier, Marc; Toulmé, Jean Jacques

    2016-01-01

    The human Matrix MetalloProtease-9 (hMMP-9) is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B), fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG) and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of 99mTc-MAG-aptamer and 111In-DOTA-F3B. 99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of 111In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, pDOTA-F3B with 2.0%ID/g than for the 111In-DOTA-control oligonucleotide (0.7%ID/g) with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor. PMID:26901393

  4. Peptide aptamers as new tools to modulate clathrin-mediated internalisation — inhibition of MT1-MMP internalisation

    Directory of Open Access Journals (Sweden)

    Ferrigno Paul

    2010-07-01

    Full Text Available Abstract Background Peptide aptamers are combinatorial protein reagents that bind to targets with a high specificity and a strong affinity thus providing a molecular tool kit for modulating the function of their targets in vivo. Results Here we report the isolation of a peptide aptamer named swiggle that interacts with the very short (21 amino acid long intracellular domain of membrane type 1-metalloproteinase (MT1-MMP, a key cell surface protease involved in numerous and crucial physiological and pathological cellular events. Expression of swiggle in mammalian cells was found to increase the cell surface expression of MT1-MMP by impairing its internalisation. Swiggle interacts with the LLY573 internalisation motif of MT1-MMP intracellular domain, thus disrupting the interaction with the μ2 subunit of the AP-2 internalisation complex required for endocytosis of the protease. Interestingly, swiggle-mediated inhibition of MT1-MMP clathrin-mediated internalisation was also found to promote MT1-MMP-mediated cell migration. Conclusions Taken together, our results provide further evidence that peptide aptamers can be used to dissect molecular events mediated by individual protein domains, in contrast to the pleiotropic effects of RNA interference techniques.

  5. 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.

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

    International Nuclear Information System (INIS)

    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

  7. Tip-induced gating of molecular levels in carbene-based junctions.

    Science.gov (United States)

    Foti, Giuseppe; Vázquez, Héctor

    2016-03-29

    We study the conductance of N-heterocyclic carbene-based (NHC) molecules on gold by means of first-principles calculations based on density-functional theory and non-equilibrium Green's functions. We consider several tip structures and find a strong dependence of the position of the NHC molecular levels with the atomistic structure of the tip. The position of the lowest unoccupied molecular orbital (LUMO) can change by almost 0.8 eV with tip shape. Through an analysis of the net charge transfer, electron redistribution and work function for each tip structure, we rationalize the LUMO shifts in terms of the sum of the work function and the maximum electrostatic potential arising from charge rearrangement. These differences in the LUMO position, effectively gating the molecular levels, result in large conductance variations. These findings open the way to modulating the conductance of NHC-based molecular circuits through the controlled design of the tip atomistic structure. PMID:26891059

  8. 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...

  9. Effects of molecular structural variants on serum Krebs von den Lungen-6 levels in sarcoidosis

    Directory of Open Access Journals (Sweden)

    Shigemura Masahiko

    2012-07-01

    Full Text Available Abstract Background Serum Krebs von den Lungen-6 (KL-6, which is classified as human mucin-1 (MUC1, is used as a marker of sarcoidosis and other interstitial lung diseases. However, there remain some limitations due to a lack of information on the factors contributing to increased levels of serum KL-6. This study was designed to investigate the factors contributing to increased levels of serum KL-6 by molecular analysis. Methods Western blot analysis using anti-KL-6 antibody was performed simultaneously on the bronchoalveolar lavage fluid (BALF and serum obtained from 128 subjects with sarcoidosis. Results KL-6/MUC1 in BALF showed three bands and five band patterns. These band patterns were associated with the MUC1 genotype and the KL-6 levels. KL-6/MUC1 band patterns in serum were dependent on molecular size class in BALF. Significantly increased levels of serum KL-6, serum/BALF KL-6 ratio and serum soluble interleukin 2 receptor were observed in the subjects with influx of high molecular size KL-6/MUC1 from the alveoli to blood circulation. The multivariate linear regression analysis involving potentially relevant variables such as age, gender, smoking status, lung parenchymal involvement based on radiographical stage and molecular size of KL-6/MUC1 in serum showed that the molecular size of KL-6/MUC1 in serum was significant independent determinant of serum KL-6 levels. Conclusions The molecular structural variants of KL-6/MUC1 and its leakage behavior affect serum levels of KL-6 in sarcoidosis. This information may assist in the interpretation of serum KL-6 levels in sarcoidosis.

  10. Prediction of organic molecular crystal geometries from MP2-level fragment quantum mechanical/molecular mechanical calculations

    Science.gov (United States)

    Nanda, Kaushik D.; Beran, Gregory J. O.

    2012-11-01

    The fragment-based hybrid many-body interaction (HMBI) model provides a computationally affordable means of applying electronic structure wavefunction methods to molecular crystals. It combines a quantum mechanical treatment of individual molecules in the unit cell and their short-range pairwise interactions with a polarizable molecular mechanics force-field treatment of long-range and many-body interactions. Here, we report the implementation of analytic nuclear gradients for the periodic model to enable full relaxation of both the atomic positions and crystal lattice parameters. Using a set of five, chemically diverse molecular crystals, we compare the quality of the HMBI MP2/aug-cc-pVDZ-level structures with those obtained from dispersion-corrected periodic density functional theory, B3LYP-D*, and from the Amoeba polarizable force field. The MP2-level structures largely agree with the experimental lattice parameters to within 2%, and the root-mean-square deviations in the atomic coordinates are less than 0.2 Å. These MP2 structures are almost as good as those predicted from periodic B3LYP-D*/TZP and are significantly better than those obtained with B3LYP-D*/6-31G(d,p) or with the Amoeba force field.

  11. Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

    OpenAIRE

    Shi, Shali; Yu, Xiaoyan; Gao, Yimin; Xue, Binbin; Wu, Xinjiao; Wang, Xiaohong; Yang, Darong; Zhu, Haizhen

    2014-01-01

    Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core...

  12. Isolation of high-affinity GTP aptamers from partially structured RNA libraries

    OpenAIRE

    DAVIS, JONATHAN H.; Szostak, Jack W.

    2002-01-01

    Aptamers, RNA sequences that bind to target ligands, are typically isolated by in vitro selection from RNA libraries containing completely random sequences. To see whether higher-affinity aptamers can be isolated from partially structured RNA libraries, we selected for aptamers that bind GTP, starting from a mixture of fully random and partially structured libraries. Because stem-loops are common motifs in previously characterized aptamers, we designed the partially structured library to cont...

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

    OpenAIRE

    Rangnekar, Abhijit; Zhang, Alex M.; Shiyuan Li, Susan; M. Bompiani, Kristin; Hansen, Majken Nørgaard; Gothelf, Kurt Vesterager; Sullenger, Bruce A; LaBean, Thomas H.

    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 a flexible single-strand linker, have been shown to possess anticoagulant activity. Here, we link multiple aptamers at programmed positions on DNA nanostructures to optimize spacing and orientation o...

  14. A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm

    OpenAIRE

    Ikebukuro, Kazunori; Okumura, Yuji; SUMIKURA, Koichi; Karube, Isao

    2005-01-01

    Thrombin-inhibiting DNA aptamers have already been obtained through the systematic evolution of ligands by exponential enrichment (SELEX). However, SELEX is a method that screens DNA aptamers that bind to their target molecules, and it sometimes fails to screen good inhibitors. Therefore, it is necessary to develop a method of screening DNA aptamers based on their inhibitory effects on the target molecules. We developed a novel method of detecting aptamers using an evolution-mimicking algorit...

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

    OpenAIRE

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

    2002-01-01

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

  16. General approach for engineering small-molecule-binding DNA split aptamers.

    Science.gov (United States)

    Kent, Alexandra D; Spiropulos, Nicholas G; Heemstra, Jennifer M

    2013-10-15

    Here we report a general method for engineering three-way junction DNA aptamers into split aptamers. Split aptamers show significant potential for use as recognition elements in biosensing applications, but reliable methods for generating these sequences are currently lacking. We hypothesize that the three-way junction is a "privileged architecture" for the elaboration of aptamers into split aptamers, as it provides two potential splitting sites that are distal from the target binding pocket. We propose a general method for split aptamer engineering that involves removing one loop region, then systematically modifying the number of base pairs in the remaining stem regions in order to achieve selective assembly only in the presence of the target small molecule. We screen putative split aptamer sequence pairs using split aptamer proximity ligation (StAPL) technology developed by our laboratory, but we validate that the results obtained using StAPL translate directly to systems in which the aptamer fragments are assembling noncovalently. We introduce four new split aptamer sequences, which triples the number of small-molecule-binding DNA split aptamers reported to date, and the methods described herein provide a reliable route for the engineering of additional split aptamers, dramatically advancing the potential substrate scope of DNA assembly based biosensors. PMID:24033257

  17. 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...

  18. A Multi-Step and Multi-Level Approach for Computer Aided Molecular Design

    DEFF Research Database (Denmark)

    Harper, Peter Mathias; Gani, Rafiqul

    . The problem formulation step incorporates a knowledge base for the identification and setup of the design criteria. Candidate compounds are identified using a multi-level generate and test CAMD solution algorithm capable of designing molecules having a high level of molecular detail. A post solution......A general multi-step approach for setting up, solving and solution analysis of computer aided molecular design (CAMD) problems is presented. The approach differs from previous work within the field of CAMD since it also addresses the need for a computer aided problem formulation and result analysis...

  19. Calculation of energy levels and wavefunctions of hydrogen molecular ion using B-splines function

    International Nuclear Information System (INIS)

    Energy levels and wavefunctions of the ground state and the first excited state of hydrogen molecular ion are calculated by solving stationary Schrodinger equation with B-splines functions. By adopting nuclear positions as knots of B-splines basis, high accuracy of energy levels of the ground state and the first excited state for hydrogen molecular ion can be reached even for the larger internuclear separations, and our ι dependent radial wavefunctions of the ground state are in a good agreement with those computed from GAUSSIAN chemistry software. (authors)

  20. A Multi-Step and Multi-Level Approach for Computer Aided Molecular Design

    DEFF Research Database (Denmark)

    Harper, Peter Mathias; Gani, Rafiqul

    A general multi-step approach for setting up, solving and solution analysis of computer aided molecular design (CAMD) problems is presented. The approach differs from previous work within the field of CAMD since it also addresses the need for a computer aided problem formulation and result analysis....... The problem formulation step incorporates a knowledge base for the identification and setup of the design criteria. Candidate compounds are identified using a multi-level generate and test CAMD solution algorithm capable of designing molecules having a high level of molecular detail. A post solution...... step for result analysis and verification is included in the methodology. (C) 2000 Elsevier Science Ltd. All rights reserved....

  1. Elevated levels of high-molecular-weight adiponectin in type 1 diabetes

    DEFF Research Database (Denmark)

    Leth, H.; Andersen, K.K.; Frystyk, J.;

    2008-01-01

    BACKGROUND: Several studies have shown that type 1 diabetic patients have elevated total levels of the adipocyte-derived adipocytokine adiponectin. However, adiponectin circulates in three different subforms, and the high-molecular-weight (HMW) subform is believed to be the primary biologically...... active form. The effects of the medium-molecular-weight (MMW) subform and the low-molecular-weight (LMW) subform are still unresolved. PURPOSE: The objective of the study was to investigate the distribution of the three molecular subforms of adiponectin in well-characterized groups of type 1 diabetics...... measured using a validated in-house time-resolved immunoflourometric assay after separation by fast protein liquid chromatography. RESULTS: The absolute concentrations of total adiponectin and all subforms were higher in type 1 diabetic patients than healthy controls. However, the relative HMW fraction was...

  2. 核酸适体(aptamer):一种具有潜力的肿瘤药物"靶向配基"%Aptamer:A potential antitumor drugs"targeted ligand"

    Institute of Scientific and Technical Information of China (English)

    郝兰; 袁耿彪; 王志刚

    2012-01-01

    核酸适体(aptamer)可描述为化学抗体,是用配体指数富集法系统进化(SELEX)技术筛选获得的单链DNA或RNA,借其自身形成的空间结构与靶标分子特异性识别,具有靶分子广、亲和力高、特异性强、易改造修饰等特点.本文简述核酸适体作为肿瘤药物"靶向配基"的应用研究.%The aptamer is described as chemical antibody- It is single DNA. or RNA. that can be isolated against by an iterative in vi1.ro process called systematic evolution of ligands by exponential enrichment (SELEX) and can identify target molecules specificity through its own space structure. They possess the molecular recognition properties in terms of their high affinity, strong specificity and easy modified. This paper describes the aptamer application research as an antitumor drug ''targeted ligands''.

  3. A Multi-step and Multi-level approach for Computer Aided Molecular Design

    DEFF Research Database (Denmark)

    A general multi-step approach for setting up, solving and solution analysis of computer aided molecular design (CAMD) problems is presented. The approach differs from previous work within the field of CAMD since it also addresses the need for a computer aided problem formulation and result analysis....... The problem formulation step incorporates a knowledge base for the identification and setup of the design criteria. Candidate compounds are identified using a multi-level generate and test CAMD solution algorithm capable of designing molecules having a high level of molecular detail. A post solution...... step using an Integrated Computer Aided System (ICAS) for result analysis and verification is included in the methodology. Keywords: CAMD, separation processes, knowledge base, molecular design, solvent selection, substitution, group contribution, property prediction, ICAS Introduction The use of...

  4. Mechanisms of molecular electronic rectification through electronic levels with strong vibrational coupling

    DEFF Research Database (Denmark)

    Kuznetsov, A.M.; Ulstrup, Jens

    2002-01-01

    We present a new view and an analytical formalism of electron flow through a donor-acceptor molecule inserted between a pair of metal electrodes. The donor and acceptor levels are strongly coupled to an environmental nuclear continuum. The formalism applies to molecular donor-acceptor systems bot...

  5. Acousto-microfluidics for screening of ssDNA aptamer

    Science.gov (United States)

    Park, Jee-Woong; Lee, Su Jin; Ren, Shuo; Lee, Sangwook; Kim, Soyoun; Laurell, Thomas

    2016-01-01

    We demonstrate a new screening method for obtaining a prostate-specific antigen (PSA) binding aptamer based on an acoustofluidic separation (acoustophoreis) technique. Since acoustophoresis provides simultaneous washing and separation in a continuous flow mode, we efficiently obtained a PSA binding aptamer that shows high affinity without any additional washing step, which is necessary in other screening methods. In addition, next-generation sequencing (NGS) was applied to accelerate the identification of the screened ssDNA pool, improving the selecting process of the aptamer candidate based on the frequency ranking of the sequences. After the 8th round of the acoustophoretic systematic evolution of ligands by exponential enrichment (SELEX) and following sequence analysis with NGS, 7 PSA binding ssDNA aptamer-candidates were obtained and characterized with surface plasmon resonance (SPR) for affinity and specificity. As a result of the new SELEX method with PSA as the model target protein, the best PSA binding aptamer showed specific binding to PSA with a dissociation constant (Kd) of 0.7 nM. PMID:27272884

  6. 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.

  7. 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)

  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. Acousto-microfluidics for screening of ssDNA aptamer.

    Science.gov (United States)

    Park, Jee-Woong; Lee, Su Jin; Ren, Shuo; Lee, Sangwook; Kim, Soyoun; Laurell, Thomas

    2016-01-01

    We demonstrate a new screening method for obtaining a prostate-specific antigen (PSA) binding aptamer based on an acoustofluidic separation (acoustophoreis) technique. Since acoustophoresis provides simultaneous washing and separation in a continuous flow mode, we efficiently obtained a PSA binding aptamer that shows high affinity without any additional washing step, which is necessary in other screening methods. In addition, next-generation sequencing (NGS) was applied to accelerate the identification of the screened ssDNA pool, improving the selecting process of the aptamer candidate based on the frequency ranking of the sequences. After the 8(th) round of the acoustophoretic systematic evolution of ligands by exponential enrichment (SELEX) and following sequence analysis with NGS, 7 PSA binding ssDNA aptamer-candidates were obtained and characterized with surface plasmon resonance (SPR) for affinity and specificity. As a result of the new SELEX method with PSA as the model target protein, the best PSA binding aptamer showed specific binding to PSA with a dissociation constant (Kd) of 0.7 nM. PMID:27272884

  10. Isolation of an Aptamer that Binds Specifically to E. coli

    Science.gov (United States)

    Cleto, Fernanda; Krieger, Marco Aurélio; Cardoso, Josiane

    2016-01-01

    Escherichia coli is a bacterial species found ubiquitously in the intestinal flora of animals, although pathogenic variants cause major public health problems. Aptamers are short oligonucleotides that bind to targets with high affinity and specificity, and have great potential for use in diagnostics and therapy. We used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to isolate four single stranded DNA (ssDNA) aptamers that bind strongly to E. coli cells (ATCC generic strain 25922), with Kd values in the nanomolar range. Fluorescently labeled aptamers label the surface of E. coli cells, as viewed by fluorescent microscopy. Specificity tests with twelve different bacterial species showed that one of the aptamers–called P12-31—is highly specific for E. coli. Importantly, this aptamer binds to Meningitis/sepsis associated E. coli (MNEC) clinical isolates, and is the first aptamer described with potential for use in the diagnosis of MNEC-borne pathologies. PMID:27104834

  11. Osteomyelitis diagnosis by 99mTc radiolabeled aptamers

    International Nuclear Information System (INIS)

    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 99mTc and used for bacteria identification in an osteomyelitis experimental model. The aptamers selected to S. aureus were directly labelled with 99mTc and were evaluated by biodistribution studies. Wistar rats with intraosseous infection in the right paw were used. A random aptamer labelled with 99mTc was as control. Six animals were used in each group. The aptamers labeled with 99mTc 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)

  12. Analytical bioconjugates, aptamers, enable specific quantitative detection of Listeria monocytogenes.

    Science.gov (United States)

    Lee, Sang-Hee; Ahn, Ji-Young; Lee, Kyeong-Ah; Um, Hyun-Ju; Sekhon, Simranjeet Singh; Sun Park, Tae; Min, Jiho; Kim, Yang-Hoon

    2015-06-15

    As a major human pathogen in the Listeria genus, Listeria monocytogenes causes the bacterial disease listeriosis, which is a serious infection caused by eating food contaminated with the bacteria. We have developed an aptamer-based sandwich assay (ABSA) platform that demonstrates a promising potential for use in pathogen detection using aptamers as analytical bioconjugates. The whole-bacteria SELEX (WB-SELEX) strategy was adopted to generate aptamers with high affinity and specificity against live L. monocytogenes. Of the 35 aptamer candidates tested, LMCA2 and LMCA26 reacted to L. monocytogenes with high binding, and were consequently chosen as sensing probes. The ABSA platform can significantly enhance the sensitivity by employing a very specific aptamer pair for the sandwich complex. The ABSA platform exhibited a linear response over a wide concentration range of L. monocytogenes from 20 to 2×10(6) CFU per mL and was closely correlated with the following relationship: y=9533.3x+1542.3 (R(2)=0.99). Our proposed ABSA platform also provided excellent specificity for the tests to distinguish L. monocytogenes from other Listeria species and other bacterial genera (3 Listeria spp., 4 Salmonella spp., 2 Vibrio spp., 3 Escherichia coli and 3 Shigella spp.). Improvements in the sensitivity and specificity have not only facilitated the reliable detection of L. monocytogenes at extremely low concentrations, but also allowed for the development of a 96-well plate-based routine assay platform for multivalent diagnostics. PMID:25590973

  13. A label free aptamer-based LPG sensor for detection of mercury in aquatic solutions

    Science.gov (United States)

    Nikbakht, Hamed; Latifi, Hamid; Ziaee, Farzaneh

    2015-09-01

    We demonstrate a label free fiber optic sensor for detection of mercury ions in aquatic solutions. This sensor utilizes aptamers as bio-recognition element which traps mercury ions and cause a refractive index change in the vicinity of the sensor. Refractive index variations lead to a change in the transmission spectrum that can be used to calculate the concentration of mercury ions in that solution. The concentration of 1 nM mercury ions was detected which is below the specific amount determined by the US environmental protection agency as the maximum authorized contaminant level of Hg2+ ions in drinking water.

  14. Improved Aptamers for the Diagnosis and Potential Treatment of HER2-Positive Cancer

    OpenAIRE

    Marlies Gijs; Gregory Penner; Garth B. Blackler; Impens, Nathalie R.E.N.; Sarah Baatout; André Luxen; Aerts, An M.

    2016-01-01

    Aptamers provide a potential source of alternative targeting molecules for existing antibody diagnostics and therapeutics. In this work, we selected novel DNA aptamers targeting the HER2 receptor by an adherent whole-cell SELEX approach. Individual aptamers were identified by next generation sequencing and bioinformatics analysis. Two aptamers, HeA2_1 and HeA2_3, were shown to bind the HER2 protein with affinities in the nanomolar range. In addition, both aptamers were able to bind with high ...

  15. Recent Progress in Aptamer-Based Functional Probes for Bioanalysis and Biomedicine.

    Science.gov (United States)

    Zhang, Huimin; Zhou, Leiji; Zhu, Zhi; Yang, Chaoyong

    2016-07-11

    Nucleic acid aptamers are short synthetic DNA or RNA sequences that can bind to a wide range of targets with high affinity and specificity. In recent years, aptamers have attracted increasing research interest due to their unique features of high binding affinity and specificity, small size, excellent chemical stability, easy chemical synthesis, facile modification, and minimal immunogenicity. These properties make aptamers ideal recognition ligands for bioanalysis, disease diagnosis, and cancer therapy. This review highlights the recent progress in aptamer selection and the latest applications of aptamer-based functional probes in the fields of bioanalysis and biomedicine. PMID:27243551

  16. Blocking interaction of viral gp120 and CD4-expressing T cells by single-stranded DNA aptamers

    OpenAIRE

    Zhao, Nianxi; Pei, Sung-nan; Parekh, Parag; Salazar, Eric; Zu, Youli

    2014-01-01

    To investigate the potential clinical application of aptamers to prevention of HIV infection, single- stranded DNA (ssDNA) aptamers specific for CD4 were developed using the systematic evolution of ligands by exponential enrichment approach and next generation sequencing. In contrast to RNA-based aptamers, the developed ssDNA aptamers were stable in human serum up to 12 hr. Cell binding assays revealed that the aptamers specifically targeted CD4-expressing cells with high binding affinity (Kd...

  17. The Selection of DNA Aptamers for Two Different Epitopes of Thrombin Was Not Due to Different Partitioning Methods

    OpenAIRE

    Wilson, Robert; Cossins, Andrew; Nicolau, Dan V.; Missailidis, Sotiris

    2013-01-01

    Nearly all aptamers identified so far for any given target molecule have been specific for the same binding site (epitope). The most notable exception to the 1 aptamer per target molecule rule is the pair of DNA aptamers that bind to different epitopes of thrombin. This communication refutes the suggestion that these aptamers exist because different partitioning methods were used when they were selected. The possibility that selection of these aptamers was biased by conflicting secondary stru...

  18. Mapping hydrophobicity on the protein molecular surface at atom-level resolution.

    Directory of Open Access Journals (Sweden)

    Dan V Nicolau

    Full Text Available A precise representation of the spatial distribution of hydrophobicity, hydrophilicity and charges on the molecular surface of proteins is critical for the understanding of the interaction with small molecules and larger systems. The representation of hydrophobicity is rarely done at atom-level, as this property is generally assigned to residues. A new methodology for the derivation of atomic hydrophobicity from any amino acid-based hydrophobicity scale was used to derive 8 sets of atomic hydrophobicities, one of which was used to generate the molecular surfaces for 35 proteins with convex structures, 5 of which, i.e., lysozyme, ribonuclease, hemoglobin, albumin and IgG, have been analyzed in more detail. Sets of the molecular surfaces of the model proteins have been constructed using spherical probes with increasingly large radii, from 1.4 to 20 Å, followed by the quantification of (i the surface hydrophobicity; (ii their respective molecular surface areas, i.e., total, hydrophilic and hydrophobic area; and (iii their relative densities, i.e., divided by the total molecular area; or specific densities, i.e., divided by property-specific area. Compared with the amino acid-based formalism, the atom-level description reveals molecular surfaces which (i present an approximately two times more hydrophilic areas; with (ii less extended, but between 2 to 5 times more intense hydrophilic patches; and (iii 3 to 20 times more extended hydrophobic areas. The hydrophobic areas are also approximately 2 times more hydrophobicity-intense. This, more pronounced "leopard skin"-like, design of the protein molecular surface has been confirmed by comparing the results for a restricted set of homologous proteins, i.e., hemoglobins diverging by only one residue (Trp37. These results suggest that the representation of hydrophobicity on the protein molecular surfaces at atom-level resolution, coupled with the probing of the molecular surface at different geometric

  19. Improved Aptamers for the Diagnosis and Potential Treatment of HER2-Positive Cancer

    Science.gov (United States)

    Gijs, Marlies; Penner, Gregory; Blackler, Garth B.; Impens, Nathalie R.E.N.; Baatout, Sarah; Luxen, André; Aerts, An M.

    2016-01-01

    Aptamers provide a potential source of alternative targeting molecules for existing antibody diagnostics and therapeutics. In this work, we selected novel DNA aptamers targeting the HER2 receptor by an adherent whole-cell SELEX approach. Individual aptamers were identified by next generation sequencing and bioinformatics analysis. Two aptamers, HeA2_1 and HeA2_3, were shown to bind the HER2 protein with affinities in the nanomolar range. In addition, both aptamers were able to bind with high specificity to HER2-overexpressing cells and HER2-positive tumor tissue samples. Furthermore, we demonstrated that aptamer HeA2_3 is being internalized into cancer cells and has an inhibitory effect on cancer cell growth and viability. In the end, we selected novel DNA aptamers with great potential for the diagnosis and possible treatment of HER2-positive cancer. PMID:27213406

  20. Improved Aptamers for the Diagnosis and Potential Treatment of HER2-Positive Cancer

    Directory of Open Access Journals (Sweden)

    Marlies Gijs

    2016-05-01

    Full Text Available Aptamers provide a potential source of alternative targeting molecules for existing antibody diagnostics and therapeutics. In this work, we selected novel DNA aptamers targeting the HER2 receptor by an adherent whole-cell SELEX approach. Individual aptamers were identified by next generation sequencing and bioinformatics analysis. Two aptamers, HeA2_1 and HeA2_3, were shown to bind the HER2 protein with affinities in the nanomolar range. In addition, both aptamers were able to bind with high specificity to HER2-overexpressing cells and HER2-positive tumor tissue samples. Furthermore, we demonstrated that aptamer HeA2_3 is being internalized into cancer cells and has an inhibitory effect on cancer cell growth and viability. In the end, we selected novel DNA aptamers with great potential for the diagnosis and possible treatment of HER2-positive cancer.

  1. 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.

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

    Science.gov (United States)

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

    2016-08-15

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

  3. Aptamer-based therapeutics and their potential in radiopharmaceutical design

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Catia S.M. [The Open University Milton Keynes, (UNited Kingdom). Chemistry Dept.; University of Toronto, Ontario (Canada). Ontario Cancer Institute; E-mail: s.missailidis@open.ac.uk; Missailidis, Sotiris [University of Toronto, Ontario (Canada). Ontario Cancer Institute

    2007-09-15

    Aptamers, short, single stranded oligonucleotide entities, have been developed in the past 15 years against a plethora of targets and for a variety of applications. These range from inhibition of receptors and enzymes to the identification of small molecules in sensor applications, and from the development of targeted therapeutic to the design of novel diagnostic and imaging agents. Furthermore, aptamers have been designed for targets that cover a wide range of diseases, from HIV to tropical diseases, cancer and inflammation. Their easy development and flexibility of use and manipulation, offers further potential. In this paper we review their selection and consider some of the recent applications of aptamers in the design of radiopharmaceuticals for the targeted radiotherapy and medical imaging of disease. (author)

  4. Oligonucleotide aptamers: A next-generation technology for the capture and detection of circulating tumor cells.

    Science.gov (United States)

    Dickey, David D; Giangrande, Paloma H

    2016-03-15

    A critical challenge for treating cancer is the early identification of those patients who are at greatest risk of developing metastatic disease. The number of circulating tumor cells (CTCs) in cancer patients has recently been shown to be a valuable (and non-invasively accessible) diagnostic indicator of the state of metastatic disease. CTCs are rare cancer cells found in the blood circulation of cancer patients believed to provide a means of diagnosing the likelihood for metastatic spread and assessing response to therapy in advanced, as well as early stage disease settings. Numerous technical efforts have been made to reliably detect and quantify CTCs, but the development of a universal assay has proven quite difficult. Notable challenges for developing a broadly useful CTC-based diagnostic assay are the development of easy-to-operate methods that (1) are sufficiently sensitive to reliably detect the small number of CTCs that are present in the circulation and (2) can capture the molecular heterogeneity of tumor cells. In this review, we describe recent progress towards the application of synthetic oligonucleotide aptamers as promising, novel, robust tools for the isolation and detection of CTCs. Advantages and challenges of the aptamer approach are also discussed. PMID:26631715

  5. 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.

  6. 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.

  7. 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.

  8. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    International Nuclear Information System (INIS)

    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 Kd 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 Kd 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

  9. Turning randomness into meaning at the molecular level using Muller's morphs

    Directory of Open Access Journals (Sweden)

    Kathleen Henson

    2012-02-01

    While evolutionary theory follows from observable facts and logical inferences (Mayr, 1985, historically, the origin of novel inheritable variations was a major obstacle to acceptance of natural selection (Bowler, 1992; Bowler, 2005. While molecular mechanisms address this issue (Jablonka and Lamb, 2005, analysis of responses to the Biological Concept Inventory (BCI (Klymkowsky et al., 2010, revealed that molecular biology majors rarely use molecular level ideas in their discourse, implying that they do not have an accessible framework within which to place evolutionary variation. We developed a “Socratic tutorial” focused on Muller's categorization of mutations' phenotypic effects (Muller, 1932. Using a novel vector-based method to analyzed students' essay responses, we found that a single interaction with this tutorial led to significant changes in thinking toward a clearer articulation of the effects of mutational change. We suggest that Muller's morphs provides an effective framework for facilitating student learning about mutational effects and evolutionary mechanisms.

  10. Selection and Biosensor Application of Aptamers for Small Molecules

    Science.gov (United States)

    Pfeiffer, Franziska; Mayer, Günter

    2016-01-01

    Small molecules play a major role in the human body and as drugs, toxins, and chemicals. Tools to detect and quantify them are therefore in high demand. This review will give an overview about aptamers interacting with small molecules and their selection. We discuss the current state of the field, including advantages as well as problems associated with their use and possible solutions to tackle these. We then discuss different kinds of small molecule aptamer-based sensors described in literature and their applications, ranging from detecting drinking water contaminations to RNA imaging. PMID:27379229

  11. A Versatile Approach Towards Nucleobase-Modified Aptamers.

    Science.gov (United States)

    Tolle, Fabian; Brändle, Gerhard M; Matzner, Daniel; Mayer, Günter

    2015-09-01

    A novel and versatile method has been developed for modular expansion of the chemical space of nucleic acid libraries, thus enabling the generation of nucleobase-modified aptamers with unprecedented recognition properties. Reintroduction of the modification after enzymatic replication gives broad access to many chemical modifications. This wide applicability, which is not limited to a single modification, will rapidly advance the application of in vitro selection approaches beyond what is currently feasible and enable the generation of aptamers to many targets that have so far not been addressable. PMID:26224087

  12. Pseudomonas viridiflava, a multi host plant pathogen with significant genetic variation at the molecular level.

    Directory of Open Access Journals (Sweden)

    Panagiotis F Sarris

    Full Text Available The pectinolytic species Pseudomonas viridiflava has a wide host range among plants, causing foliar and stem necrotic lesions and basal stem and root rots. However, little is known about the molecular evolution of this species. In this study we investigated the intraspecies genetic variation of P. viridiflava amongst local (Cretan, as well as international isolates of the pathogen. The genetic and phenotypic variability were investigated by molecular fingerprinting (rep-PCR and partial sequencing of three housekeeping genes (gyrB, rpoD and rpoB, and by biochemical and pathogenicity profiling. The biochemical tests and pathogenicity profiling did not reveal any variability among the isolates studied. However, the molecular fingerprinting patterns and housekeeping gene sequences clearly differentiated them. In a broader phylogenetic comparison of housekeeping gene sequences deposited in GenBank, significant genetic variability at the molecular level was found between isolates of P. viridiflava originated from different host species as well as among isolates from the same host. Our results provide a basis for more comprehensive understanding of the biology, sources and shifts in genetic diversity and evolution of P. viridiflava populations and should support the development of molecular identification tools and epidemiological studies in diseases caused by this species.

  13. Molecular-level engineering of protein physical hydrogels for predictive sol-gel phase behavior

    OpenAIRE

    Mulyasasmita, Widya; Lee, Ji Seok; Heilshorn, Sarah C.

    2011-01-01

    Predictable tuning of bulk mechanics from the molecular level remains elusive in many physical hydrogel systems due to the reliance on non-specific and non-stoichiometric chain interactions for network formation. We describe a Mixing-Induced Two-Component Hydrogel (MITCH) system, in which network assembly is driven by specific and stoichiometric peptide-peptide binding interactions. By integrating protein science methodologies with simple polymer physics model, we manipulate the polypeptide b...

  14. Effect of heparin and low-molecular weight heparin on serum potassium and sodium levels

    OpenAIRE

    Girish M Bengalorkar; N Sarala; Venkatrathnamma, P. N.; Kumar, T. N.

    2011-01-01

    Introduction: To study the effects of heparin and low-molecular weight heparin (LMWH) on potassium and sodium levels in patients with cardiovascular diseases (CVDs) and stroke. Materials and Methods : Sixty patients were recruited with 30 patients each receiving heparin and enoxaparin. Patients with CVD and stroke receiving heparin and LMWH were compared for their demographic profile and laboratory data, and this was analyzed by descriptive statistics. Risk factors associated with the develop...

  15. Sources of genetic resistance in maize to Fusarium stalk rot andtheir variations at molecular level

    OpenAIRE

    QURESHI, SAJJAD HUSSAIN; Qayyum, Abdul; FIERS, WILL

    2015-01-01

    Identifying the resistant genotypes is one of the vital strategies to control Fusarium stalk rot disease in maize. Fifty accessions of maize germplasm were evaluated for resistance to stalk rot caused by Fusarium verticillioides at the Maize and Millet Research Institute, Yousafwala, Pakistan, during the spring and autumn of 2010, and their genetic variations were also studied at the molecular level to avoid environmental effects in the Department of Medicinal Chemistry, University of Minneso...

  16. Investigation of variables influencing cognitive inhibition: from the behavioral to the molecular level

    OpenAIRE

    Dieler, Alica Christina

    2011-01-01

    The present work investigated the neural mechanisms underlying cognitive inhibition/thought suppression in Anderson’s and Green’s Think/No-Think paradigm (TNT), as well as different variables influencing these mechanisms at the cognitive, the neurophysiological, the electrophysiological and the molecular level. Neurophysiological data collected with fNIRS and fMRI have added up to the existing evidence of a fronto-hippocampal network interacting during the inhibition of unwanted thoughts. Som...

  17. Sensing, physiological effects and molecular response to elevated CO2 levels in eukaryotes

    OpenAIRE

    Sharabi, Kfir; Lecuona, Emilia; Helenius, Iiro Taneli; Beitel, Greg J.; Sznajder, Jacob Iasha; Gruenbaum, Yosef

    2009-01-01

    Carbon dioxide (CO2) is an important gaseous molecule that maintains biosphere homeostasis and is an important cellular signalling molecule in all organisms. The transport of CO2 through membranes has fundamental roles in most basic aspects of life in both plants and animals. There is a growing interest in understanding how CO2 is transported into cells, how it is sensed by neurons and other cell types and in understanding the physiological and molecular consequences of elevated CO2 levels (h...

  18. Application of Aptamers in Food Safety%核酸适体技术在食品安全分析中的应用

    Institute of Scientific and Technical Information of China (English)

    徐敦明; 吴敏; 邹远; 张强; 吴崔晨; 周昱; 刘贤进

    2011-01-01

    Aptamers are all new ligands with a high affinity for considerably differing molecules ranging from largetargets as proteins over peptides, complex molecules to drugs and organic small molecules or even metal ions. These molecules are identified and selected through an in vitro process called systematic evolution of ligands by exponential enrichment (SELEX). Aptamers are widely used, including medical and pharmaceutical basic research, drug development, diagnosis, and therapy. Analytical and separation tools bearing aptamers as molecular recognition and binding elements are another big field of application. The SELEX method was modified over the years in different ways to become more efficient and less time consuming, to reach higher affinities of aptamers selected and higher automation of process. In reviewing this technology, the article covers the topic, following a general introduction, under the headings: the SELEX protocol, the development of aptamers by use of SELEX, and the application of aptamers in food safety.%核酸适体(Aptamer)是经体外筛选技术(SELEX)筛选出的能特异结合蛋白质或其它小分子物质的寡聚核苷酸片段,对可结合的配体有严格的识别能力和高度的亲和力.经过十几年的发展,SELEX技术已经成为一种重要的研究手段和工具,被广泛应用到分子生物学、基因组学、临床医学等领域.高通量筛选的技术特点与Aptamer精确识别、易体外合成与修饰等特性,使得Aptamer在分析化学与生物医药研究方面具有广阔的应用前景.本文对提高筛选效率和效果为目标的核酸适体筛选技术新进展、核酸适体技术在食品安全分析中的应用进行了回顾,展望了核酸适体在食品安全分析上的应用前景.

  19. Molecular-Level Processes Governing the Interaction of Contaminants with Iron and Manganese Oxides - Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Brown Jr., G. E.; Chambers, S. A.

    1999-10-31

    Many of the inorganic and organic contaminants present in sediments at DOE sites can be altered or destroyed by reduction and oxidation (redox) reactions occurring at mineral surfaces. A fundamental understanding of such redox processes provided by molecular-level studies on structurally and compositionally well-defined mineral surfaces will lead to: (i) improved models of contaminant fate and transport in geochemical systems, and (ii) optimized manipulation of these processes for remediation purposes. To contribute to this understanding, we will study, both experimentally and theoretically, redox processes involving three important contaminants - chromate ion, carbon tetrachloride, and trichloroethene TCE, on the following iron and manganese oxides - hematite, magnetite, maghemite, and pyrolusite. These oxides and their hydroxylated analogs commonly occur as coatings on minerals or as interfaces in the subsurface environment. Single-crystal surfaces of these oxides will be synthesized in carefully controlled fashion by molecular beam epitaxy. These surfaces, as well as high surface are powdered samples of these oxides, will be used in spectroscopic and kinetic experiments in both aqueous and gas phases. Our goal is to identify products and to determine the kinetics and mechanisms of surface-catalyzed redox reaction of Cr(VI) and CR(III), and the reductive dechlorination of carbon tetrachloride and TCE. The combination of theory and experiment will provide the base information needed to scale from the molecular level to the microscopic grain level minerals.

  20. Effects of molecular structural variants on serum Krebs von den Lungen-6 levels in sarcoidosis

    OpenAIRE

    Shigemura Masahiko; Nasuhara Yasuyuki; Konno Satoshi; Shimizu Chikara; Matsuno Kazuhiko; Yamguchi Etsuro; Nishimura Masaharu

    2012-01-01

    Abstract Background Serum Krebs von den Lungen-6 (KL-6), which is classified as human mucin-1 (MUC1), is used as a marker of sarcoidosis and other interstitial lung diseases. However, there remain some limitations due to a lack of information on the factors contributing to increased levels of serum KL-6. This study was designed to investigate the factors contributing to increased levels of serum KL-6 by molecular analysis. Methods Western blot analysis using anti-KL-6 antibody was performed s...

  1. Effects of molecular structural variants on serum Krebs von den Lungen-6 levels in sarcoidosis

    OpenAIRE

    Shigemura, Masahiko; Nasuhara, Yasuyuki; Konno, Satoshi; Shimizu, Chikara; Matsuno, Kazuhiko; Yamguchi, Etsuro; Nishimura, Masaharu

    2012-01-01

    Background: Serum Krebs von den Lungen-6 (KL-6), which is classified as human mucin-1 (MUC1), is used as a marker of sarcoidosis and other interstitial lung diseases. However, there remain some limitations due to a lack of information on the factors contributing to increased levels of serum KL-6. This study was designed to investigate the factors contributing to increased levels of serum KL-6 by molecular analysis. Methods: Western blot analysis using anti-KL-6 antibody was performed simultan...

  2. A Highlight of Recent Advances in Aptamer Technology and Its Application

    Directory of Open Access Journals (Sweden)

    Hongguang Sun

    2015-06-01

    Full Text Available Aptamers and SELEX (systematic evolution of ligands by exponential enrichment technology have gained increasing attention over the past 25 years. Despite their functional similarity to protein antibodies, oligonucleotide aptamers have many unique properties that are suitable for clinical applications and industrialization. Aptamers may be superior to antibodies in fields such as biomarker discovery, in vitro and in vivo diagnosis, precisely controlled drug release, and targeted therapy. However, aptamer commercialization has not occurred as quickly as expected, and few aptamer-based products have yet successfully entered clinical and industrial use. Thus, it is important to critically review some technical barriers of aptamer and SELEX technology per se that may impede aptamer development and application. To date, how to rapidly obtain aptamers with superior bioavailability over antibodies remains the key issue. In this review, we discuss different chemical and structural modification strategies aimed to enhance aptamer bioavailability. We also discuss improvements to SELEX process steps to shorten the selection period and improve the SELEX process success rate. Applications in which aptamers are particularly suited and perform differently or superior to antibodies are briefly introduced.

  3. Inhibition of hepatitis C virus infection by DNA aptamer against envelope protein.

    Science.gov (United States)

    Yang, Darong; Meng, Xianghe; Yu, Qinqin; Xu, Li; Long, Ying; Liu, Bin; Fang, Xiaohong; Zhu, Haizhen

    2013-10-01

    Hepatitis C virus (HCV) envelope protein (E1E2) is essential for virus binding to host cells. Aptamers have been demonstrated to have strong promising applications in drug development. In the current study, a cDNA fragment encoding the entire E1E2 gene of HCV was cloned. E1E2 protein was expressed and purified. Aptamers for E1E2 were selected by the method of selective evolution of ligands by exponential enrichment (SELEX), and the antiviral actions of the aptamers were examined. The mechanism of their antiviral activity was investigated. The data show that selected aptamers for E1E2 specifically recognize the recombinant E1E2 protein and E1E2 protein from HCV-infected cells. CD81 protein blocks the binding of aptamer E1E2-6 to E1E2 protein. Aptamers against E1E2 inhibit HCV infection in an infectious cell culture system although they have no effect on HCV replication in a replicon cell line. Beta interferon (IFN-β) and IFN-stimulated genes (ISGs) are not induced in virus-infected hepatocytes with aptamer treatment, suggesting that E1E2-specific aptamers do not induce innate immunity. E2 protein is essential for the inhibition of HCV infection by aptamer E1E2-6, and the aptamer binding sites are located in E2. Q412R within E1E2 is the major resistance substitution identified. The data indicate that an aptamer against E1E2 exerts its antiviral effects through inhibition of virus binding to host cells. Aptamers against E1E2 can be used with envelope protein to understand the mechanisms of HCV entry and fusion. The aptamers may hold promise for development as therapeutic drugs for hepatitis C patients. PMID:23877701

  4. 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. PMID:27494117

  5. Aptamer based, non-PCR, non-serological detection of Chagas disease biomarkers in Trypanosoma cruzi infected mice.

    Directory of Open Access Journals (Sweden)

    Rana Nagarkatti

    Full Text Available Chagas disease affects about 5 million people across the world. The etiological agent, the intracellular parasite Trypanosoma cruzi (T. cruzi, can be diagnosed using microscopy, serology or PCR based assays. However, each of these methods has their limitations regarding sensitivity and specificity, and thus to complement these existing diagnostic methods, alternate assays need to be developed. It is well documented that several parasite proteins called T. cruzi Excreted Secreted Antigens (TESA, are released into the blood of an infected host. These circulating parasite antigens could thus be used as highly specific biomarkers of T. cruzi infection. In this study, we have demonstrated that, using a SELEx based approach, parasite specific ligands called aptamers, can be used to detect TESA in the plasma of T. cruzi infected mice. An Enzyme Linked Aptamer (ELA assay, similar to ELISA, was developed using biotinylated aptamers to demonstrate that these RNA ligands could interact with parasite targets. Aptamer L44 (Apt-L44 showed significant and specific binding to TESA as well as T. cruzi trypomastigote extract and not to host proteins or proteins of Leishmania donovani, a related trypanosomatid parasite. Our result also demonstrated that the target of Apt-L44 is conserved in three different strains of T. cruzi. In mice infected with T. cruzi, Apt-L44 demonstrated a significantly higher level of binding compared to non-infected mice suggesting that it could detect a biomarker of T. cruzi infection. Additionally, Apt-L44 could detect these circulating biomarkers in both the acute phase, from 7 to 28 days post infection, and in the chronic phase, from 55 to 230 days post infection. Our results show that Apt-L44 could thus be used in a qualitative ELA assay to detect biomarkers of Chagas disease.

  6. Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device.

    Science.gov (United States)

    Sanghavi, Bankim J; Moore, John A; Chávez, Jorge L; Hagen, Joshua A; Kelley-Loughnane, Nancy; Chou, Chia-Fu; Swami, Nathan S

    2016-04-15

    Monitoring the periodic diurnal variations in cortisol from small volume samples of serum or saliva is of great interest, due to the regulatory role of cortisol within various physiological functions and stress symptoms. Current detection assays are immunologically based and require cumbersome antibody immobilization chemistries, thereby limiting the assay versatility, kinetics, and reproducibility. We present a quantitative aptamer-based detection methodology for cortisol that does not require target labeling, capture probe immobilization on the detection surface or wash steps prior to readout. Using a recognition system of aptamer functionalized gold nanoparticles pre-bound with electro-active triamcinolone, the cortisol level is detected based on its competitive binding to the aptamer by following signal from the displaced triamcinolone using square wave voltammetry at patterned graphene-modified electrodes in a microfluidic or nanoslit device. Due to the 3D analyte diffusion profile at the aptamer interface and the ability to enhance the surface area for cortisol capture, this assay shows signal linearity over a five-log analyte concentration range (10 μg/mL to 30 pg/mL) and exhibits rapid binding kinetics with cortisol versus other glucocorticoids, as apparent from the absence of interferences from estradiol, testosterone and progesterone. The assay is carried out within the biologically relevant range for glucocorticoids in serum and saliva matrices, and benchmarked versus ELISA and radioimmunoassays. Based on absence of cumbersome surface immobilization and wash steps for carrying out this assay, its quantitative signal characteristics and its ability to resist interferences from other glucocorticoids, we envision its application towards routine monitoring of cortisol within bio-fluids. PMID:26618642

  7. APTAMER CAPTURE AND OPTICAL INTERFEROMETRIC DETECTION OF CYANOBACTERIAL TOXINS

    Science.gov (United States)

    Cyanobacterial toxins have been identified as a health risk in source and finished waters passing through drinking water utilities in the United States. In this project, a rapid, sensitive and field usable sensor based on an aptamer modified planar waveguide interferometric se...

  8. Quantum Dot- and Aptamer-Based Nanostructures for Biological Applications

    Science.gov (United States)

    Meshik, Xenia

    Quantum dots are semiconductor nanoparticles that have gained popularity in optical and electronic applications in recent years. Aptamers are short man-made oligonucleotides with high binding affinity for a specific target. One part of this work presents an optical FRET-based sensor for K+ and Pb2+ consisting of a fluorescent quantum dot, an aptamer, and a gold nanoparticle quencher. Additionally, an electrochemical sensor for K+ and Pb2+ is also presented, which consists of an aptamer with an electron donor bound to graphene. Both sensors are shown to detect K+ and Pb2+ at concentrations critical for human health. The emission spectrum of the optical sensor is also shown to shift in response to strong electric fields. UV-excited TiO 2 quantum dots are also investigated for their ability to influence the dynamics of voltage gated ion channels in cells. It was found that the activation voltage is shifted in the presence of UV-excited TiO2 quantum dots. Electrostatic force measurements and theoretical calculations confirm that electric fields in TiO2 can in fact be optically induced. ZnO quantum dots are also synthesized and their optical and electrical properties are similarly investigated. Additionally, Raman and surface-enhanced Raman spectroscopy is used in this work to find previously-unknown spectra of the aptamer Apt-alphavbeta3 and the peptide thymosin-beta4.

  9. The thrombin binding aptamer GGTTGGTGTGGTTGG forms a bimolecular guanine tetraplex

    Czech Academy of Sciences Publication Activity Database

    Fialová, Markéta; Kypr, Jaroslav; Vorlíčková, Michaela

    2006-01-01

    Roč. 344, 1 (2006), s. 50-54. ISSN 0006-291X R&D Projects: GA AV ČR(CZ) IAA4004201 Institutional research plan: CEZ:AV0Z50040507 Keywords : DNA tetraplex * thrombin binding aptamer * CD spectroscopy Subject RIV: BO - Biophysics Impact factor: 2.855, year: 2006

  10. 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. PMID:27427591

  11. Colorimetric logic response based on aptamer functionalized colloidal crystal hydrogels

    Science.gov (United States)

    Ye, Baofen; Wang, Huan; Ding, Haibo; Zhao, Yuanjin; Pu, Yuepu; Gu, Zhongze

    2015-04-01

    A novel colorimetric logic system based on the aptamer-cross-linked colloidal crystal hydrogel (CCH) was developed. With the input stimuli of Hg2+ and Ag+, the CCH displayed shrinking response and colour change corresponding to the logical ``OR'' and ``AND'' gate. The visualization of the logic output signals is realized.A novel colorimetric logic system based on the aptamer-cross-linked colloidal crystal hydrogel (CCH) was developed. With the input stimuli of Hg2+ and Ag+, the CCH displayed shrinking response and colour change corresponding to the logical ``OR'' and ``AND'' gate. The visualization of the logic output signals is realized. Electronic supplementary information (ESI) available: I. Experimental section. II. Photograph of the aptamer functionalized CCH in the presence of different targets. III. The specificity of the aptamer functionalized CCH. IV. Relationship between the input ion concentration and the reflection wavelength blue shift. V. The logic swelling kinetics of CCH. See DOI: 10.1039/c5nr00586h

  12. 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.

  13. Aptamer/Graphene Quantum Dots Nanocomposite Capped Fluorescent Mesoporous Silica Nanoparticles for Intracellular Drug Delivery and Real-Time Monitoring of Drug Release.

    Science.gov (United States)

    Zheng, Fen-Fen; Zhang, Peng-Hui; Xi, Yu; Chen, Jing-Jia; Li, Ling-Ling; Zhu, Jun-Jie

    2015-12-01

    Great challenges in investigating the release of drug in complex cellular microenvironments necessitate the development of stimuli-responsive drug delivery systems with real-time monitoring capability. In this work, a smart drug nanocarrier based on fluorescence resonance energy transfer (FRET) is fabricated by capping graphene quantum dots (GQDs, the acceptor) onto fluorescent mesoporous silica nanoparticles (FMSNs, the donor) via ATP aptamer for real-time monitoring of ATP-triggered drug release. Under extracellular conditions, the fluorescence of FMSNs remains in the "off" state in the low ATP level which is unable to trigger the release of drug. Once specifically recognized and internalized into the target tumor cells by AS1411 aptamer, in the ATP-rich cytoplasm, the conformation switch of the ATP aptamer causes the shedding of the GQDs from the nanocarriers, leading to the release of the loaded drugs and consequently severe cytotoxicity. Simultaneously, the fluorescence of FMSNs turns "on" along with the dissociation of GQDs, which allows real-time monitoring of the release of drug from the pores. Such a drug delivery system features high specificity of dual-target recognition with AS1411 and ATP aptamer as well as high sensitivity of the FRET-based monitoring strategy. Thus, the proposed multifunctional ATP triggered FRET-nanocarriers will find potential applications for versatile drug-release monitoring, efficient drug transport, and targeted cancer therapeutics. PMID:26524192

  14. Triplex molecular beacons for sensitive recognition of melamine based on abasic-site-containing DNA and fluorescent silver nanoclusters.

    Science.gov (United States)

    Wang, Ya; Sun, Qianqian; Zhu, Linling; Zhang, Junying; Wang, Fengyang; Lu, Linlin; Yu, Haijun; Xu, Zhiai; Zhang, Wen

    2015-05-01

    A melamine aptamer derived from an abasic-site-containing triplex molecular beacon (tMB) was designed and developed for sensitive recognition of melamine by integrating tMBs and fluorescent silver nanoclusters (Ag NCs). PMID:25865656

  15. Coexistence of spinodal instability and thermal nucleation in thin-film rupture: Insights from molecular levels

    Science.gov (United States)

    Nguyen, Trung Dac; Fuentes-Cabrera, Miguel; Fowlkes, Jason D.; Rack, Philip D.

    2014-03-01

    Despite extensive investigation using hydrodynamic models and experiments over the past decades, there remain open questions regarding the origin of the initial rupture of thin liquid films. One of the reasons that makes it difficult to identify the rupture origin is the coexistence of two dewetting mechanisms, namely, thermal nucleation and spinodal instability, as observed in many experimental studies. Using a coarse-grained model and large-scale molecular dynamics simulations, we are able to characterize the very early stage of dewetting in nanometer-thick liquid-metal films wetting a solid substrate. We observe the features characteristic of both spinodal instability and thermal nucleation in the spontaneously dewetting films and show that these two macroscopic mechanisms share a common origin at molecular levels.

  16. 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.

  17. 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...

  18. Evaluation of Phonon-level Density of UO2 by Molecular Dynamics Simulation

    Institute of Scientific and Technical Information of China (English)

    Hui-fen Zhang; Gan Li; Xiao-feng Tian; Tao Gao

    2011-01-01

    Molecular dynamics calculation of UO2 in a wide temperature range are presented and discussed.The calculated lattice parameters,mean square displacements,and dynamic property of phonon-level density of the velocity auto-correlation functions for UO2 are provided.The Morelon potential and the Basak potential are employed.It confirms that the calculated lattice parameters using the Basak potential are in nearly perfect agreement with the reported values.The models successfully predict mean square displacement and Bredig transition.Furthermore,the phonon-level density of uranium dioxide are discussed.The intensity of phonon-level density increases with temperature,and the properties of UO2 are characterized by large thermal vibrations rather than extensive disorder.

  19. 全细胞的核酸适配体筛选的研究进展%Research advances of aptamer selection for whole cell

    Institute of Scientific and Technical Information of China (English)

    刘品多; 屈锋

    2016-01-01

    核酸适配体(aptamer)是从人工合成的随机单链 DNA(ssDNA)或 RNA 文库中筛选得到的,能够高亲和力、高特异性地与靶标结合的 ssDNA或 RNA。核酸适配体的靶标范围广,可包括小分子、蛋白质、细胞、微生物等多种靶标。其中以细胞为靶标的适配体在生物感应、分子成像、医学诊断、药物传输和疾病治疗等领域有很大的应用潜能。但全细胞的核酸适配体筛选过程复杂,筛选难度大,筛选的适配体性能不佳是导致目前可用的适配体非常有限的主要原因。由于细胞表面蛋白质在提取纯化过程中分子结构和形态会发生改变,故以膜表面蛋白质为靶标筛选的适配体很难应用于识别整体细胞。以全细胞为靶标的核酸适配体筛选则不需要准确了解细胞表面的分子结构,筛选过程中可保持细胞的天然状态,以全细胞为靶标筛选出的核酸适配体有望直接用于全细胞识别。本文总结了2008~2015年全细胞的核酸适配体筛选的研究进展,介绍了靶细胞的分类、核酸库的设计、筛选条件和方法以及核酸适配体的亲和力表征方法等。并列出全细胞靶标的核酸适配体序列。%Aptamers are single stranded DNA( ssDNA)or RNA that may bind to small mole-cules,proteins,cells,microorganisms,and other targets. Typically,aptamers are generated by a selection process referred to as systematic evolution of ligands by exponential enrichment ( SELEX). Aptamers that bind with high affinity and specificity to proteins that reside on the cell surface have potential utility in the fields of biosenser,molecular imaging,medical diagno-sis,drug delivery,and disease treatment. However,till now,available aptamers are very limit-ed mainly due to the complex and difficulty screening of aptamer and their poor properties. Using purified cell surface proteins as target has the disadvantage of changing structure. When target

  20. Aptaligner: Automated Software for Aligning Pseudorandom DNA X-Aptamers from Next-Generation Sequencing Data

    OpenAIRE

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

    2014-01-01

    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 provid...

  1. Predicting the Uncertain Future of Aptamer-Based Diagnostics and Therapeutics

    OpenAIRE

    Bruno, John G.

    2015-01-01

    Despite the great promise of nucleic acid aptamers in the areas of diagnostics and therapeutics for their facile in vitro development, lack of immunogenicity and other desirable properties, few truly successful aptamer-based products exist in the clinical or other markets. Core reasons for these commercial deficiencies probably stem from industrial commitment to antibodies including a huge financial investment in humanized monoclonal antibodies and a general ignorance about aptamers and their...

  2. Anti-peptide aptamers recognize amino acid sequence and bind a protein epitope.

    OpenAIRE

    Xu, W; Ellington, A. D.

    1996-01-01

    In vitro selection of nucleic acid binding species (aptamers) is superficially similar to the immune response. Both processes produce biopolymers that can recognize targets with high affinity and specificity. While antibodies are known to recognize the sequence and conformation of protein surface features (epitopes), very little is known about the precise interactions between aptamers and their epitopes. Therefore, aptamers that could recognize a particular epitope, a peptide fragment of huma...

  3. ESCORT APTAMERS: NEW TOOLS FOR THE TARGETEDDELIVERY OF THERAPEUTICS INTO CELLS

    OpenAIRE

    Davydova, A.; Vorobjeva, M.; Venyaminova, A.

    2011-01-01

    Escort aptamers are DNA or RNA sequences with high affinity to certain cell-surface proteins, which can be used for targeted delivery of various agents into cells of a definite type. The peculiarities of the selection of escort aptamers are discussed in this review. The methods used in selection of escort aptamers via the SELEX technique are considered, including selection against isolated cell-surface proteins, cell fragments, living eukaryotic cells, and bacteria. Particular attention is gi...

  4. Labelling of live cells using fluorescent aptamers: binding reversal with DNA nucleases

    OpenAIRE

    Terazono Hideyuki; Anzai Yu; Soloviev Mikhail; Yasuda Kenji

    2010-01-01

    Abstract A reversible cell labelling method has been developed for non-destructive and non-invasive cell labelling and purification. Our method uses high affinity single strand DNA (ssDNA) aptamers against surface exposed target molecules on cells. The aptamers are subsequently removed from the cell surface using DNase nuclease treatment. We exemplified our method by labelling human acute lymphoblastic leukemia cells with Qdot-ssDNA aptamers, and restoring them to the label-free condition by ...

  5. DNA Aptamers against Taiwan Banded Krait α-Bungarotoxin Recognize Taiwan Cobra Cardiotoxins

    OpenAIRE

    Ying-Jung Chen; Chia-Yu Tsai; Wan-Ping Hu; Long-Sen Chang

    2016-01-01

    Bungarus multicinctus α-bungarotoxin (α-Bgt) and Naja atra cardiotoxins (CTXs) share a common structural scaffold, and their tertiary structures adopt three-fingered loop motifs. Four DNA aptamers against α-Bgt have been reported previously. Given that the binding of aptamers with targeted proteins depends on structural complementarity, in this study, we investigated whether DNA aptamers against α-Bgt could also recognize CTXs. It was found that N. atra cardiotoxin 3 (CTX3) reduced the electr...

  6. Light-up properties of complexes between thiazole orange-small molecule conjugates and aptamers

    OpenAIRE

    Pei, Renjun; Rothman, Jeffrey; Xie, Yuli; Stojanovic, Milan N.

    2009-01-01

    The full understanding of dynamics of cellular processes hinges on the development of efficient and non-invasive labels for intracellular RNA species. Light-up aptamers binding fluorogenic ligands show promise as specific labels for RNA species containing those aptamers. Herein, we took advantage of existing, non-light-up aptamers against small molecules and demonstrated a new class of light-up probes in vitro. We synthesized two conjugates of thiazole orange dye to small molecules (GMP and A...

  7. 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.

  8. Aptamers: An in vitro Evolution of Therapeutic and Diagnostic Applications in Medicine

    OpenAIRE

    Kayhan, Basak; Uner KAYABAs

    2013-01-01

    Aptamers are nucleic acid oligomers with distinct conformational shapes that allow binding targets with high affinity and specificity. Selective Evolution of Ligands by Exponential Enrichment (SELEX); an in vitro selection process to develop aptamers, has been invented in 1990. Despite more than 20 years have passed after its discovery, products of SELEX technology are in use in medicine. In this review we discuss why we need aptamers not only in therapeutic but also in diagnostic application...

  9. 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 intersting 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. PMID:26984473

  10. Inhibition of hepatitis C virus production by aptamers against the core protein.

    Science.gov (United States)

    Shi, Shali; Yu, Xiaoyan; Gao, Yimin; Xue, Binbin; Wu, Xinjiao; Wang, Xiaohong; Yang, Darong; Zhu, Haizhen

    2014-02-01

    Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-β) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C. PMID:24307579

  11. Chimeric nucleolin aptamer with survivin DNAzyme for cancer cell targeted delivery.

    Science.gov (United States)

    Subramanian, Nithya; Kanwar, Jagat R; Akilandeswari, Balachandran; Kanwar, Rupinder K; Khetan, Vikas; Krishnakumar, Subramanian

    2015-04-25

    A chimeric aptamer-DNAzyme conjugate was generated for the first time using a nucleolin aptamer (NCL-APT) and survivin Dz (Sur_Dz) and exhibited the targeted killing of cancer cells. This proof of concept of using an aptamer for the delivery of DNAzyme can be applied to other cancer types to target survivin in cancer cells in a specific manner. PMID:25797393

  12. Effect of heparin and low-molecular weight heparin on serum potassium and sodium levels

    Directory of Open Access Journals (Sweden)

    Girish M Bengalorkar

    2011-01-01

    Full Text Available Introduction: To study the effects of heparin and low-molecular weight heparin (LMWH on potassium and sodium levels in patients with cardiovascular diseases (CVDs and stroke. Materials and Methods : Sixty patients were recruited with 30 patients each receiving heparin and enoxaparin. Patients with CVD and stroke receiving heparin and LMWH were compared for their demographic profile and laboratory data, and this was analyzed by descriptive statistics. Risk factors associated with the development of hyperkalemia were analyzed using multiple logistic regression model. Results : There was an increase in potassium levels and decrease in sodium levels compared with baseline in both the groups. The difference between the groups with respect to sodium and potassium levels was not statistically significant. On analysis, the risk factors for development of hyperkalemia were baseline potassium levels, serum creatinine, and creatinine clearance. The change in sodium and potassium levels on the fifth day of therapy was increased with LMWH compared with heparin, although not statistically significant. Conclusions : The clinician should anticipate hyperkalemia especially in patients with renal impairment receiving these drugs.

  13. Molecular MRI differentiation of VEGF receptor-2 levels in C6 and RG2 glioma models.

    Science.gov (United States)

    He, Ting; Smith, Nataliya; Saunders, Debra; Pittman, Benjamin P; Lerner, Megan; Lightfoot, Stanley; Silasi-Mansat, Robert; Lupu, Florea; Towner, Rheal A

    2013-01-01

    Vascular endothelial growth factor receptor 2 (VEGFR2) is an important angiogenic marker over-expressed in gliomas. With the use of molecular magnetic resonance imaging (mMRI) differing levels of VEGFR2 can be characterized in vivo with in rodent gliomas varying in angiogenesis. VEGFR2 levels were assessed by intravenous administration of an anti-VEGFR2 probe (anti-VEGFR2-albumin-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin) into C6 or RG2 glioma-bearing rats, and visualized with mMRI. A non-specific IgG was coupled to the albumin-Gd-DTPA-biotin construct as a contrast agent molecular weight control. VEGFR2 levels are heterogeneous in different regions of C6 gliomas, whereas VEGFR2 was more homogenous or evenly distributed in RG2 gliomas. RG2 gliomas have less VEGFR2 within tumor periphery and peri-necrotic (pmMRI results were confirmed with fluorescence staining and mean fluorescence intensity (MFI) quantification of the anti-VEGFR2 probe in excised glioma and brain tissues, as well as detection of VEGFR2 in C6 and RG2 gliomas and corresponding contalateral brain tissues. Ex vivo VEGFR2 levels were found to be significantly higher in C6 gliomas compared to RG2 tumors (p<0.001), which corresponded with in vivo detection using the VEGFR2 probe. Immunohistochemistry staining for HIF-1α (hypoxia inducible factor 1α), which is associated with angiogenesis, indicated higher levels in RG2 (p<0.01) compared to C6 gliomas. The data suggests that C6 gliomas have angiogenesis which is associated more with large blood vessels in tumor periphery and peri-necrotic regions, and less microvascular angiogenesis within the tumor interior, compared to RG2 gliomas. PMID:23901356

  14. One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

    Directory of Open Access Journals (Sweden)

    Stöcklein Walter

    2007-08-01

    Full Text Available Abstract Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

  15. Improvement of a streptavidin-binding aptamer by LNA- and α-l-LNA-substitutions

    DEFF Research Database (Denmark)

    Jørgensen, Anna S; Hansen, Lykke H; Vester, Birte;

    2014-01-01

    Forty modified versions of a streptavidin-binding aptamer each containing single or multiple LNA or α-l-LNA-substitutions were synthesized and their dissociation constants determined by surface plasmon resonance experiments. Both full-length and truncated versions of the aptamer were studied and...... compared with the unmodified DNA aptamers. A ∼two-fold improvement in binding affinity was achieved by incorporation of LNA nucleotides in the 3'-part of the stems of the streptavidin-binding aptamer whereas LNA- and α-l-LNA-substitutions in the terminal stem increased the serum stability....

  16. Inhibition of hepatitis C virus infection by NS5A-specific aptamer.

    Science.gov (United States)

    Yu, Xiaoyan; Gao, Yimin; Xue, Binbin; Wang, Xiaohong; Yang, Darong; Qin, Yuwen; Yu, Rong; Liu, Nianli; Xu, Li; Fang, Xiaohong; Zhu, Haizhen

    2014-06-01

    To increase efficacy of hepatitis C treatment, future regiments will incorporate multiple direct-acting antiviral drugs. HCV NS5A protein was expressed and purified. Aptamers against NS5A were screened and obtained by the selective evolution of ligands by exponential enrichment approach and the antiviral actions of the aptamers were tested. The mechanisms through which the aptamers exert their antiviral activity were explored. The aptamers NS5A-4 and NS5A-5 inhibit HCV RNA replication and infectious virus production without causing cytotoxicity in human hepatocytes. The aptamers do not affect hepatitis B virus replication in HepG2.2.15 cells. Interferon beta (IFN-β) and interferon-stimulated genes (ISGs) are not induced by the aptamers in HCV-infected hepatocytes. Further study shows that domain I and domain III of NS5A protein are involved in the suppression of HCV RNA replication and infectious virus production by NS5A-4. Y2105H within NS5A is the major resistance mutation identified. NS5A aptamer disrupts the interaction of NS5A with core protein. The data suggest that the aptamers against NS5A protein may exert antiviral effects through inhibiting viral RNA replication, preventing the interaction of NS5A with core protein. Aptamers for NS5A may be used to understand the mechanisms of virus replication and assembly and served as potential therapeutic agents for hepatitis C. PMID:24713119

  17. 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.

  18. 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

    2016-12-01

    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. PMID:27299653

  19. Development of structure switching aptamer assay for detection of aflatoxin M1 in milk sample.

    Science.gov (United States)

    Sharma, Atul; Catanante, Gaëlle; Hayat, Akhtar; Istamboulie, Georges; Ben Rejeb, Ines; Bhand, Sunil; Marty, Jean Louis

    2016-09-01

    The discovery of in-vitro systematic evolution of ligands by exponential enrichment (SELEX) process has considerably broaden the utility of aptamer as bio-recognition element, providing the high binding affinity and specificity against the target analytes. Recent research has focused on the development of structure switching signaling aptamer assay, transducing the aptamer- target recognition event into an easily detectable signal. In this paper, we demonstrate the development of structure switching aptamer assay for determination of aflatoxin M1 (AFM1) employing the quenching-dequenching mechanism. Hybridization of fluorescein labelled anti-AFM1 aptamer (F-aptamer) with TAMRA labelled complementary sequences (Q-aptamer) brings the fluorophore and the quencher into close proximity, which results in maximum fluorescence quenching. On addition of AFM1, the target induced conformational formation of antiparallel G-quadruplex aptamer-AFM1 complex results in fluorescence recovery. Under optimized experimental conditions, the developed method showed the good linearity with limit of detection (LOD) at 5.0ngkg(-1) for AFM1. The specificity of the sensing platform was carefully investigated against aflatoxin B1 (AFB1) and ochratoxin A (OTA). The developed assay platform showed the high specificity towards AFM1. The practical application of the developed aptamer assay was verified for detection of AFM1 in spiked milk samples. Good recoveries were obtained in the range from 94.40% to 95.28% (n=3) from AFM1 spiked milk sample. PMID:27343575

  20. Short-Term Dynamics of North Sea Bacterioplankton-Dissolved Organic Matter Coherence on Molecular Level.

    Science.gov (United States)

    Lucas, Judith; Koester, Irina; Wichels, Antje; Niggemann, Jutta; Dittmar, Thorsten; Callies, Ulrich; Wiltshire, Karen H; Gerdts, Gunnar

    2016-01-01

    Remineralization and transformation of dissolved organic matter (DOM) by marine microbes shape the DOM composition and thus, have large impact on global carbon and nutrient cycling. However, information on bacterioplankton-DOM interactions on a molecular level is limited. We examined the variation of bacterial community composition (BCC) at Helgoland Roads (North Sea) in relation to variation of molecular DOM composition and various environmental parameters on short-time scales. Surface water samples were taken daily over a period of 20 days. Bacterial community and molecular DOM composition were assessed via 16S rRNA gene tag sequencing and ultrahigh resolution Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), respectively. Environmental conditions were driven by a coastal water influx during the first half of the sampling period and the onset of a summer phytoplankton bloom toward the end of the sampling period. These phenomena led to a distinct grouping of bacterial communities and DOM composition which was particularly influenced by total dissolved nitrogen (TDN) concentration, temperature, and salinity, as revealed by distance-based linear regression analyses. Bacterioplankton-DOM interaction was demonstrated in strong correlations between specific bacterial taxa and particular DOM molecules, thus, suggesting potential specialization on particular substrates. We propose that a combination of high resolution techniques, as used in this study, may provide substantial information on substrate generalists and specialists and thus, contribute to prediction of BCC variation. PMID:27014241

  1. Study on the mechanism of action between dimethyl phthalate and herring sperm DNA at molecular level.

    Science.gov (United States)

    Chi, Zhenxing; Wang, Donglin; You, Hong

    2016-08-01

    Dimethyl phthalate (DMP), a typical phthalic acid ester, is widespread in the environment and causes extensive concern due to its adverse effects on human health. To understand the genotoxicity of DMP at molecular level, the toxic interaction of DMP with herring sperm (hs) deoxyribonucleic acid (DNA; hs-DNA) was investigated in vitro under simulated physiological conditions using multi-spectroscopic techniques and a molecular modeling method. The results of Ultraviolet-Visible absorption spectroscopy, fluorescence emission spectroscopy, and circular dichroism spectra indicated that DMP interacts with hs-DNA in a groove-binding mode that changes the double helical structure of DNA. The binding constant and the number of binding sites calculated from the fluorescence quenching data were 565.718 L mol(-1) and 0.7872, respectively. A molecular modeling study revealed that DMP tends to bind with DNA in the A-T-rich regions of minor groove and that hydrogen bonding and van der Waals forces play main roles in the interaction. This research can help to elucidate the mechanism of DMP toxicity in vivo. PMID:27166703

  2. Short-term dynamics of North Sea bacterioplankton-dissolved organic matter coherence on molecular level

    Directory of Open Access Journals (Sweden)

    Judith eLucas

    2016-03-01

    Full Text Available Remineralisation and transformation of dissolved organic matter (DOM by marine microbes shape the DOM composition and thus, have large impact on global carbon and nutrient cycling. However, information on bacterioplankton-DOM interactions on a molecular level is limited. We examined the variation of bacterial community composition at Helgoland Roads (North Sea in relation to variation of molecular DOM composition and various environmental parameters on short-time scales. Surface water samples were taken daily over a period of twenty days. Bacterial community and molecular DOM composition were assessed via 16S rRNA gene tag sequencing and ultrahigh resolution Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS, respectively. Environmental conditions were driven by a coastal water influx during the first half of the sampling period and the onset of a summer phytoplankton bloom towards the end of the sampling period. These phenomena led to a distinct grouping of bacterial communities and DOM composition which was particularly influenced by total dissolved nitrogen concentration, temperature and salinity, as revealed by distance-based linear regression analyses. Bacterioplankton-DOM interaction was demonstrated in strong correlations between specific bacterial taxa and particular DOM molecules, thus, suggesting potential specialization on particular substrates. We propose that a combination of high resolution techniques, as used in this study, may provide substantial information on substrate generalists and specialists and thus, contribute to prediction of bacterial community composition variation.

  3. Core level regulatory network of osteoblast as molecular mechanism for osteoporosis and treatment

    Science.gov (United States)

    Zhu, Xiaomei; Li, Jun; Liang, Yuhong; Liu, Tao; Zhu, Yanxia; Zhang, Bingbing; Tan, Shuang; Guo, Huajie; Guan, Shuguang; Ao, Ping; Zhou, Guangqian

    2016-01-01

    To develop and evaluate the long-term prophylactic treatment for chronic diseases such as osteoporosis requires a clear view of mechanism at the molecular and systems level. While molecular signaling pathway studies for osteoporosis are extensive, a unifying mechanism is missing. In this work, we provide experimental and systems-biology evidences that a tightly connected top-level regulatory network may exist, which governs the normal and osteoporotic phenotypes of osteoblast. Specifically, we constructed a hub-like interaction network from well-documented cross-talks among estrogens, glucocorticoids, retinoic acids, peroxisome proliferator-activated receptor, vitamin D receptor and calcium-signaling pathways. The network was verified with transmission electron microscopy and gene expression profiling for bone tissues of ovariectomized (OVX) rats before and after strontium gluconate (GluSr) treatment. Based on both the network structure and the experimental data, the dynamical modeling predicts calcium and glucocorticoids signaling pathways as targets for GluSr treatment. Modeling results further reveal that in the context of missing estrogen signaling, the GluSr treated state may be an outcome that is closest to the healthy state. PMID:26783964

  4. Comparison of Whole-Cell SELEX Methods for the Identification of Staphylococcus Aureus-Specific DNA Aptamers

    OpenAIRE

    Jihea Moon; Giyoung Kim; Saet Byeol Park; Jongguk Lim; Changyeun Mo

    2015-01-01

    Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, s...

  5. Studies on liposomes with Chlorophyll for monitoring the electromagnetic influence at molecular level

    International Nuclear Information System (INIS)

    The liposomes with Chlorophyll are excellent model membranes and could be successfully used to study the electromagnetic influence at molecular level. The strong visible absorption and fluorescence of Chlorophyll allow its use as sensor for the interactions at molecular level and as a fluorescence marker; it reflects certain aspects of the supramolecular structure of the lipid phase: fluidity, lipid and liposomes aggregation. The objective of our work was to evidence athermal effect of low level, pulsed microwave (MW) fields on liposomes and to evidence the possible mechanism of interaction at molecular level. Unilamellar liposomes were obtained from multilamellar vesicles by the hand-shaken method and sonication for 30 minutes. The multilamellar vesicles were prepared using Chla /lipid films with specific molar ratio (lipid/Chla 1/10 and 1/100) and different lipids (Dipalmitoyl phosphatidylcholine, Dimirystoyl Phosphatidylcholine and Dioleoyl Phosphatidylcholine-Sigma). The films were dispersed in buffer solutions of different pH (6.2 - 7.6). The Chlorophyll was freshly extracted from spinach leaves and separated by the chromatographic method. Portions of liposome suspension (0.6 ml) were inserted into Teflon cuvettes. The samples were irradiated in series, for periods of 5-30 minutes. The exposure system was: MW generator + adapted load (shortened rectangular waveguide) + Teflon cuvette filled with sample liquid. The effect of MW irradiation is not observable on multilamellar vesicles, but only on small unilamellar vesicles. The MW effect is athermal, verified by conventional heating in the same range of temperatures and results in enlarging the size of vesicles. The enlarging effect of MW is opposed to the effect of ultrasounds exposure. It is not clear if effects due to MW are proportional with exposure duration; it seems that this mostly depends on the type of lipid in vesicles. The UV and VIS spectra were recorded to observe the oxidation state of the

  6. Cu-Au alloy nanostructures coated with aptamers: a simple, stable and highly effective platform for in vivo cancer theranostics

    Science.gov (United States)

    Ye, Xiaosheng; Shi, Hui; He, Xiaoxiao; Yu, Yanru; He, Dinggeng; Tang, Jinlu; Lei, Yanli; Wang, Kemin

    2016-01-01

    As a star material in cancer theranostics, photoresponsive gold (Au) nanostructures may still have drawbacks, such as low thermal conductivity, irradiation-induced melting effect and high cost. To solve the problem, copper (Cu) with a much higher thermal conductivity and lower cost was introduced to generate a novel Cu-Au alloy nanostructure produced by a simple, gentle and one-pot synthetic method. Having the good qualities of both Cu and Au, the irregularly-shaped Cu-Au alloy nanostructures showed several advantages over traditional Au nanorods, including a broad and intense near-infrared (NIR) absorption band from 400 to 1100 nm, an excellent heating performance under laser irradiation at different wavelengths and even a notable photostability against melting. Then, via a simple conjugation of fluorophore-labeled aptamers on the Cu-Au alloy nanostructures, active targeting and signal output were simultaneously introduced, thus constructing a theranostic platform based on fluorophore-labeled, aptamer-coated Cu-Au alloy nanostructures. By using human leukemia CCRF-CEM cancer and Cy5-labeled aptamer Sgc8c (Cy5-Sgc8c) as the model, a selective fluorescence imaging and NIR photothermal therapy was successfully realized for both in vitro cancer cells and in vivo tumor tissues. It was revealed that Cy5-Sgc8c-coated Cu-Au alloy nanostructures were not only capable of robust target recognition and stable signal output for molecular imaging in complex biological systems, but also killed target cancer cells in mice with only five minutes of 980 nm irradiation. The platform was found to be simple, stable, biocompatible and highly effective, and shows great potential as a versatile tool for cancer theranostics.As a star material in cancer theranostics, photoresponsive gold (Au) nanostructures may still have drawbacks, such as low thermal conductivity, irradiation-induced melting effect and high cost. To solve the problem, copper (Cu) with a much higher thermal conductivity

  7. 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.

  8. Rupture of DNA aptamer: New insights from simulations

    International Nuclear Information System (INIS)

    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

  9. 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.

  10. 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 ...

  11. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

    OpenAIRE

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

    2016-01-01

    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 ...

  12. Aptamer Microarrays—Current Status and Future Prospects

    OpenAIRE

    Martin Witt; Johanna-Gabriela Walter; Frank Stahl

    2015-01-01

    Microarray technologies are state of the art in biological research, which requires fast genome, proteome and transcriptome analysis technologies. Often antibodies are applied in protein microarrays as proteomic tools. Since the generation of antibodies against toxic targets or small molecules including organic compounds remains challenging the use of antibodies may be limited in this context. In contrast to this, aptamer microarrays provide alternative techniques to circumvent these limitati...

  13. 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.

  14. Oxidized phosphatidylcholines in membrane-level cellular signaling: from biophysics to physiology and molecular pathology.

    Science.gov (United States)

    Volinsky, Roman; Kinnunen, Paavo K J

    2013-06-01

    The oxidation of lipids has been shown to impact virtually all cellular processes. The paradigm has been that this involvement is due to interference with the functions of membrane-associated proteins. It is only recently that methodological advances in molecular-level detection and identification have begun to provide insights into oxidative lipid modification and its involvement in cell signaling as well as in major diseases and inflammation. Extensive evidence suggests a correlation between lipid peroxidation and degenerative neurological diseases such as Parkinson's and Alzheimer's, as well as type 2 diabetes and cancer. Despite the obvious relevance of understanding the molecular basis of the above ailments, the exact modes of action of oxidized lipids have remained elusive. In this minireview, we summarize recent findings on the biophysical characteristics of biomembranes following oxidative derivatization of their lipids, and how these altered properties are involved in both physiological processes and major pathological conditions. Lipid-bearing, oxidatively truncated and functionalized acyl chains are known to modify membrane bulk physical properties, such as thermal phase behavior, bilayer thickness, hydration and polarity profiles, as manifest in the altered structural dynamics of lipid bilayers, leading to augmented membrane permeability, fast lipid transbilayer diffusion (flip-flop), loss of lipid asymmetry (scrambling) and phase segregation (the formation of 'rafts'). These changes, together with the generated reactive lipid derivatives, can be further expected to interfere with lipid-protein interactions, influencing metabolic pathways, causing inflammation, the execution phase in apoptosis and initiating pathological processes. PMID:23506295

  15. Organization of lipids in the tear film: a molecular-level view.

    Directory of Open Access Journals (Sweden)

    Alicja Wizert

    Full Text Available Biophysical properties of the tear film lipid layer are studied at the molecular level employing coarse grain molecular dynamics (MD simulations with a realistic model of the human tear film. In this model, polar lipids are chosen to reflect the current knowledge on the lipidome of the tear film whereas typical Meibomian-origin lipids are included in the thick non-polar lipids subphase. Simulation conditions mimic those experienced by the real human tear film during blinks. Namely, thermodynamic equilibrium simulations at different lateral compressions are performed to model varying surface pressure, and the dynamics of the system during a blink is studied by non-equilibrium MD simulations. Polar lipids separate their non-polar counterparts from water by forming a monomolecular layer whereas the non-polar molecules establish a thick outermost lipid layer. Under lateral compression, the polar layer undulates and a sorting of polar lipids occurs. Moreover, formation of three-dimensional aggregates of polar lipids in both non-polar and water subphases is observed. We suggest that these three-dimensional structures are abundant under dynamic conditions caused by the action of eye lids and that they act as reservoirs of polar lipids, thus increasing stability of the tear film.

  16. Axial level-dependent molecular and cellular mechanisms underlying the genesis of the embryonic neural plate.

    Science.gov (United States)

    Kondoh, Hisato; Takada, Shinji; Takemoto, Tatsuya

    2016-06-01

    The transcription factor gene Sox2, centrally involved in neural primordial regulation, is activated by many enhancers. During the early stages of embryonic development, Sox2 is regulated by the enhancers N2 and N1 in the anterior neural plate (ANP) and posterior neural plate (PNP), respectively. This differential use of the enhancers reflects distinct regulatory mechanisms underlying the genesis of ANP and PNP. The ANP develops directly from the epiblast, triggered by nodal signal inhibition, and via the combined action of TFs SOX2, OTX2, POU3F1, and ZIC2, which promotes the the ANP development and inhibits other cell lineages. In contrast, the PNP is derived from neuromesodermal bipotential axial stem cells that develop into the neural plate when Sox2 is activated by the N1 enhancer, whereas they develop into the paraxial mesoderm when the N1 enhancer is repressed by the action of TBX6. The axial stem cells are maintained by the activity of WNT3a and T (Brachyury). However, at axial levels more anterior to the 8th somites (cervical levels), the development of both the neural plate and somite proceeds in the absence of WNT3a, T, or TBX6. These observations indicate that distinct molecular and cellular mechanisms determine neural plate genesis based on the axial level, and contradict the classical concept of the term "neural induction," which assumes a pan-neural plate mechanism. PMID:27279156

  17. Highly Multiplexed RNA Aptamer Selection using a Microplate-based Microcolumn Device.

    Science.gov (United States)

    Reinholt, Sarah J; Ozer, Abdullah; Lis, John T; Craighead, Harold G

    2016-01-01

    We describe a multiplexed RNA aptamer selection to 19 different targets simultaneously using a microcolumn-based device, MEDUSA (Microplate-based Enrichment Device Used for the Selection of Aptamers), as well as a modified selection process, that significantly reduce the time and reagents needed for selections. We exploited MEDUSA's reconfigurable design between parallel and serially-connected microcolumns to enable the use of just 2 aliquots of starting library, and its 96-well microplate compatibility to enable the continued use of high-throughput techniques in downstream processes. Our modified selection protocol allowed us to perform the equivalent of a 10-cycle selection in the time it takes for 4 traditional selection cycles. Several aptamers were discovered with nanomolar dissociation constants. Furthermore, aptamers were identified that not only bound with high affinity, but also acted as inhibitors to significantly reduce the activity of their target protein, mouse decapping exoribonuclease (DXO). The aptamers resisted DXO's exoribonuclease activity, and in studies monitoring DXO's degradation of a 30-nucleotide substrate, less than 1 μM of aptamer demonstrated significant inhibition of DXO activity. This aptamer selection method using MEDUSA helps to overcome some of the major challenges with traditional aptamer selections, and provides a platform for high-throughput selections that lends itself to process automation. PMID:27432610

  18. Aptamer optical biosensor without bio-breakage using upconversion nanoparticles as donors

    NARCIS (Netherlands)

    K. Song; X. Kong; X. Liu; Y. Zhang; Q. Zeng; L. Tu; Z. Shi; H. Zhang

    2012-01-01

    LRET-based optical biosensor of an aptamer-upconversion conjugate was constructed. It is demonstrated that photosensitized breakage and damage of aptamers are eliminated by employing UCNPs as donors, and the as-designed biosensor is specific and sensitive in the detection of ATP.

  19. 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...

  20. AFBI assay - Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells.

    Science.gov (United States)

    Thiel, William H; Giangrande, Paloma H

    2016-07-01

    The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization. PMID:26972784

  1. A reliable aptamer array prepared by repeating inkjet-spotting toward on-site measurement.

    Science.gov (United States)

    Inoue, Suzuyo; Seyama, Michiko; Miura, Toru; Horiuchi, Tsutomu; Iwasaki, Yuzuru; Takahashi, Jun-Ichi; Hayashi, Katsuyoshi; Tamechika, Emi

    2016-11-15

    A preparation protocol is proposed for a reliable aptamer array utilizing an ink-jet spotter. We accumulated streptavidin and biotinylated-aptamer in this order on a biotinylated-polyethylene glycol-coated gold substrate to prepare an aptamer array. The aptamer array was prepared with an alternate spotting structure where each aptamer spot was placed between reference spots formed with blocking solution thus suppressing contamination from neighboring spots during the blocking and washing processes. Four aptamer spots were prepared in a small area of 1×4.8mm(2) with five reference spots made of blocking solution. We evaluated the thrombin binding ability of the spotted aptamer array using a multi-analysis surface plasmon resonance sensor. We prepared a disposable capillary-driven flow chip designed for on-site measurement (Miura et al., 2010) with our aptamer array and detected thrombin from phosphate-buffered saline at concentrations of 50ngmL(-1) and 1μgmL(-1) (equivalent to 1.35 and 27nM, respectively). A correlation was observed between the refractive index shift and thrombin concentration. This implies that our array preparation protocol meets the requirement for the preparation of a one-time-use chip for on-site measurement. PMID:27315520

  2. 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. PMID:26763942

  3. 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

    Aptamers are valuable tools that provide great potential to develop cost-effective diagnostics and therapies in the biomedical field. Here, we report a novel DNA aptamer that folds into an unconventional G-quadruplex structure able to recognize and enter specifically into human Burkitt's lymphoma...

  4. Efficient Reverse Transcription Using Locked Nucleic Acid Nucleotides towards the Evolution of Nuclease Resistant RNA Aptamers

    DEFF Research Database (Denmark)

    Crouzier, Lucile; Dubois, Camille; Edwards, Stacey L;

    2012-01-01

    Modified nucleotides are increasingly being utilized in the de novo selection of aptamers for enhancing their drug-like character and abolishing the need for time consuming trial-and-error based post-selection modifications. Locked nucleic acid (LNA) is one of the most prominent and successful...... step is a pre-requisite for performing LNA-modified RNA aptamer selection....

  5. Enzyme-linked small-molecule detection using split aptamer ligation.

    Science.gov (United States)

    Sharma, Ashwani K; Kent, Alexandra D; Heemstra, Jennifer M

    2012-07-17

    Here we report an aptamer-based analogue of the widely used sandwich enzyme-linked immunosorbent assay (ELISA). This assay utilizes the cocaine split aptamer, which is comprised of two DNA strands that only assemble in the presence of the target small molecule. One split aptamer fragment is immobilized on a microplate, then a test sample is added containing the second split aptamer fragment. If cocaine is present in the test sample, it directs assembly of the split aptamer and promotes a chemical ligation between azide and cyclooctyne functional groups appended to the termini of the split aptamer fragments. Ligation results in covalent attachment of biotin to the microplate and provides a colorimetric output upon conjugation to streptavidin-horseradish peroxidase. Using this assay, we demonstrate detection of cocaine at concentrations of 100 nM-100 μM in buffer and 1-100 μM human blood serum. The detection limit of 1 μM in serum represents an improvement of two orders of magnitude over previously reported split aptamer-based sensors and highlights the utility of covalently trapping split aptamer assembly events. PMID:22715870

  6. Oligonucleotide Functionalised Microbeads: Indispensable Tools for High-Throughput Aptamer Selection

    Directory of Open Access Journals (Sweden)

    Lewis A. Fraser

    2015-12-01

    Full Text Available The functionalisation of microbeads with oligonucleotides has become an indispensable technique for high-throughput aptamer selection in SELEX protocols. In addition to simplifying the separation of binding and non-binding aptamer candidates, microbeads have facilitated the integration of other technologies such as emulsion PCR (ePCR and Fluorescence Activated Cell Sorting (FACS to high-throughput selection techniques. Within these systems, monoclonal aptamer microbeads can be individually generated and assayed to assess aptamer candidate fitness thereby helping eliminate stochastic effects which are common to classical SELEX techniques. Such techniques have given rise to aptamers with 1000 times greater binding affinities when compared to traditional SELEX. Another emerging technique is Fluorescence Activated Droplet Sorting (FADS whereby selection does not rely on binding capture allowing evolution of a greater diversity of aptamer properties such as fluorescence or enzymatic activity. Within this review we explore examples and applications of oligonucleotide functionalised microbeads in aptamer selection and reflect upon new opportunities arising for aptamer science.

  7. Molecular-Level Simulations of Shock Generation and Propagation in Soda-Lime Glass

    Science.gov (United States)

    Grujicic, M.; Bell, W. C.; Pandurangan, B.; Cheeseman, B. A.; Fountzoulas, C.; Patel, P.

    2012-08-01

    A non-equilibrium molecular dynamics method is employed to study the mechanical response of soda-lime glass (a material commonly used in transparent armor applications) when subjected to the loading conditions associated with the generation and propagation of planar shock waves. Specific attention is given to the identification and characterization of various (inelastic-deformation and energy-dissipation) molecular-level phenomena and processes taking place at, or in the vicinity of, the shock front. The results obtained revealed that the shock loading causes a 2-4% (shock strength-dependent) density increase. In addition, an increase in the average coordination number of the silicon atoms is observed along with the creation of smaller Si-O rings. These processes are associated with substantial energy absorption and dissipation and are believed to greatly influence the blast/ballistic impact mitigation potential of soda-lime glass. The present work was also aimed at the determination of the shock Hugoniot (i.e., a set of axial stress vs. density/specific-volume vs. internal energy vs. particle velocity vs. temperature) material states obtained in soda-lime glass after the passage of a shock wave of a given strength (as quantified by the shock speed). The availability of a shock Hugoniot is critical for construction of a high deformation-rate, large-strain, high pressure material model which can be used within a continuum-level computational analysis to capture the response of a soda-lime glass based laminated transparent armor structure (e.g., a military vehicle windshield, door window, etc.) to blast/ballistic impact loading.

  8. RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug

    Directory of Open Access Journals (Sweden)

    Mamidala Praveen

    2012-01-01

    Full Text Available Abstract Background Bed bugs (Cimex lectularius are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance. Results We performed a next-generation RNA sequencing (RNA-Seq experiment to find differentially expressed genes between pesticide-resistant (PR and pesticide-susceptible (PS strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs and our previous 454 pyrosequenced database (21,088 ESTs. The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2 revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid. Conclusions We developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide

  9. Crystallization and preliminary X-ray analysis of the complex of human α-thrombin with a modified thrombin-binding aptamer

    International Nuclear Information System (INIS)

    The complex between human α-thrombin and a modified thrombin-binding aptamer has been crystallized. Diffraction data were collected to 2.15 Å resolution, the structure was solved by molecular replacement and refinement of the model is in progress. The thrombin-binding aptamer (TBA) is a consensus DNA 15-mer that binds specifically to human α-thrombin at nanomolar concentrations and inhibits its procoagulant functions. Recently, a modified TBA (mTBA) containing a 5′–5′ inversion-of-polarity site has been shown to be more stable and to possess a higher thrombin affinity than its unmodified counterpart. The structure of the thrombin–TBA complex has previously been determined at low resolution, but did not provide a detailed picture of the aptamer conformation or of the protein–DNA assembly, while that of the complex with mTBA is unknown. Crystallographic analysis of the thrombin–mTBA complex has been attempted. The crystals diffracted to 2.15 Å resolution and belonged to space group I222

  10. Detection and Characterization of Cancer Cells and Pathogenic Bacteria Using Aptamer-Based Nano-Conjugates

    Directory of Open Access Journals (Sweden)

    Vinayakumar Gedi

    2014-09-01

    Full Text Available Detection and characterization of cells using aptamers and aptamer-conjugated nanoprobes has evolved a great deal over the past few decades. This evolution has been driven by the easy selection of aptamers via in vitro cell-SELEX, permitting sensitive discrimination between target and normal cells, which includes pathogenic prokaryotic and cancerous eukaryotic cells. Additionally, when the aptamer-based strategies are used in conjunction with nanomaterials, there is the potential for cell targeting and therapeutic effects with improved specificity and sensitivity. Here we review recent advances in aptamer-based nano-conjugates and their applications for detecting cancer cells and pathogenic bacteria. The multidisciplinary research utilized in this field will play an increasingly significant role in clinical medicine and drug discovery.

  11. Application of aptamers to environmental analysis%核酸适配体在环境分析中的应用∗

    Institute of Scientific and Technical Information of China (English)

    陈慧甜; 孙清; 时国庆

    2015-01-01

    核酸适配体( aptamer)是一段体外获得的可与相应的靶分子特异性结合的寡核苷酸序列。由于其相较于抗体有易合成、易修饰、靶分子范围广等优点,近些年来在环境分析中受到广泛关注。本文简要介绍了核酸适配体的结构特点及常用筛选方法,着重按照靶标分子的不同,综述了核酸适配体在环境微生物及化合物分析检测中的应用。并就核酸适配体作为分子识别元件的优势及局限进行了分析,对未来研究进行了展望。%Aptamers are short oligonucleotides with high affinity to their targets obtained in vitro. They have attracted great attention in environmental analysis because of the advantages compared to antibodies such as easierto synthesize, modify and wider range of targets. This paper briefly described the structure characters of aptamers and its common screening methods, and also summarized the application of aptamers on the analysis of environmental microbiology and compounds according to different targets. In addition, this paper analyzed the advantage and limitations that utilizing aptamers as molecular recognition elements and discussed its future research.

  12. A combination of positive dielectrophoresis driven on-line enrichment and aptamer-fluorescent silica nanoparticle label for rapid and sensitive detection of Staphylococcus aureus.

    Science.gov (United States)

    Shangguan, Jingfang; Li, Yuhong; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Zou, Zhen; Shi, Hui

    2015-07-01

    Staphylococcus aureus (S. aureus) is an important human pathogen that causes several diseases ranging from superficial skin infections to life-threatening diseases. Here, a method combining positive dielectrophoresis (pDEP) driven on-line enrichment and aptamer-fluorescent silica nanoparticle label has been developed for the rapid and sensitive detection of S. aureus in microfluidic channels. An aptamer, having high affinity to S. aureus, is used as the molecular recognition tool and immobilized onto chloropropyl functionalized fluorescent silica nanoparticles through a click chemistry approach to obtain S. aureus aptamer-nanoparticle bioconjugates (Apt(S.aureus)/FNPs). The pDEP driven on-line enrichment technology was used for accumulating the Apt(S.aureus)/FNP labeled S. aureus. After incubating with S. aureus, the mixture of Apt(S.aureus)/FNP labeled S. aureus and Apt(S.aureus)/FNPs was directly introduced into the pDEP-based microfluidic system. By applying an AC voltage in a pDEP frequency region, the Apt(S.aureus)/FNP labelled S. aureus moved to the electrodes and accumulated in the electrode gap, while the free Apt(S.aureus)/FNPs flowed away. The signal that came from the Apt(S.aureus)/FNP labelled S. aureus in the focused detection areas was then detected. Profiting from the specificity of aptamer, signal amplification of FNP label and pDEP on-line enrichment, this assay can detect as low as 93 and 270 cfu mL(-1)S. aureus in deionized water and spiked water samples, respectively, with higher sensitivities than our previously reported Apt(S.aureus)/FNP based flow cytometry. Moreover, without the need for separation and washing steps usually required for FNP label involved bioassays, the total assay time including sample pretreatment was within 2 h. PMID:25963028

  13. Synthesis of AS1411-aptamer-conjugated CdTe quantum dots with high fluorescence strength for probe labeling tumor cells.

    Science.gov (United States)

    Alibolandi, Mona; Abnous, Khalil; Ramezani, Mohammad; Hosseinkhani, Hossein; Hadizadeh, Farzin

    2014-09-01

    In this paper, we report microwave-assisted, one-stage synthesis of high-quality functionalized water-soluble cadmium telluride (CdTe) quantum dots (QDs). By selecting sodium tellurite as the Te source, cadmium chloride as the Cd source, mercaptosuccinic acid (MSA) as the capping agent, and a borate-acetic acid buffer solution with a pH range of 5-8, CdTe nanocrystals with four colors (blue to orange) were conveniently prepared at 100 °C under microwave irradiation in less than one hour (reaction time: 10-60 min). The influence of parameters such as the pH, Cd:Te molar ratio, and reaction time on the emission range and quantum yield percentage (QY%) was investigated. The structures and compositions of the prepared CdTe QDs were characterized by transmission electron microscopy, energy-dispersive X-ray spectroscopy, selective area electron diffraction, and X-ray powder diffraction experiments. The formation mechanism of the QDs is discussed in this paper. Furthermore, AS1141-aptamer-conjugated CdTe QDs in the U87MG glioblastoma cell line were assessed with a fluorescence microscope. The obtained results showed that the best conditions for obtaining a high QY of approximately 87% are a pH of 6, a Cd:Te molar ratio of 5:1, and a 30-min reaction time at 100 °C under microwave irradiation. The results showed that AS1141-aptamer-conjugated CdTe QDs could enter tumor cells efficiently. It could be concluded that a facile high-fluorescence-strength QD conjugated with a DNA aptamer, AS1411, which can recognize the extracellular matrix protein nucleolin, can specifically target U87MG human glioblastoma cells. The qualified AS1411-aptamer-conjugated QDs prepared in this study showed excellent capabilities as nanoprobes for cancer targeting and molecular imaging. PMID:25172439

  14. Aptamer conjugated paclitaxel and magnetic fluid loaded fluorescently tagged PLGA nanoparticles for targeted cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Aravind, Athulya; Nair, Remya; Raveendran, Sreejith; Veeranarayanan, Srivani; Nagaoka, Yutaka; Fukuda, Takahiro; Hasumura, Takahashi; Morimoto, Hisao; Yoshida, Yasuhiko; Maekawa, Toru; Sakthi Kumar, D., E-mail: sakthi@toyo.jp

    2013-10-15

    Controlled and targeted drug delivery is an essential criterion in cancer therapy to reduce the side effects caused by non-specific drug release and toxicity. Targeted chemotherapy, sustained drug release and optical imaging have been achieved using a multifunctional nanocarrier constructed from poly (D, L-lactide-co-glycolide) nanoparticles (PLGA NPs), an anticancer drug paclitaxel (PTX), a fluorescent dye Nile red (NR), magnetic fluid (MF) and aptamers (Apt, AS1411, anti-nucleolin aptamer). The magnetic fluid and paclitaxel loaded fluorescently labeled PLGA NPs (MF-PTX-NR-PLGA NPs) were synthesized by a single-emulsion technique/solvent evaporation method using a chemical cross linker bis (sulfosuccinimidyl) suberate (BS3) to enable binding of aptamer on to the surface of the nanoparticles. Targeting aptamers were then introduced to the particles through the reaction with the cross linker to target the nucleolin receptors over expressed on the cancer cell surface. Specific binding and uptake of the aptamer conjugated magnetic fluid loaded fluorescently tagged PLGA NPs (Apt-MF-NR-PLGA NPs) to the target cancer cells induced by aptamers was observed using confocal microscopy. Cytotoxicity assay conducted in two cell lines (L929 and MCF-7) confirmed that targeted MCF-7 cancer cells were killed while control cells were unharmed. In addition, aptamer mediated delivery resulting in enhanced binding and uptake to the target cancer cells exhibited increased therapeutic effect of the drug. Moreover, these aptamer conjugated magnetic polymer vehicles apart from actively transporting drugs into specifically targeted tumor regions can also be used to induce hyperthermia or for facilitating magnetic guiding of particles to the tumor regions. - Highlights: • Aptamer escorted, theranostic biodegradable PLGA carriers were developed. • Can target cancer cells, control drug release, image and magnetically guide. • Highly specific to the targeted cancer cells thus delivering

  15. Aptamer conjugated paclitaxel and magnetic fluid loaded fluorescently tagged PLGA nanoparticles for targeted cancer therapy

    International Nuclear Information System (INIS)

    Controlled and targeted drug delivery is an essential criterion in cancer therapy to reduce the side effects caused by non-specific drug release and toxicity. Targeted chemotherapy, sustained drug release and optical imaging have been achieved using a multifunctional nanocarrier constructed from poly (D, L-lactide-co-glycolide) nanoparticles (PLGA NPs), an anticancer drug paclitaxel (PTX), a fluorescent dye Nile red (NR), magnetic fluid (MF) and aptamers (Apt, AS1411, anti-nucleolin aptamer). The magnetic fluid and paclitaxel loaded fluorescently labeled PLGA NPs (MF-PTX-NR-PLGA NPs) were synthesized by a single-emulsion technique/solvent evaporation method using a chemical cross linker bis (sulfosuccinimidyl) suberate (BS3) to enable binding of aptamer on to the surface of the nanoparticles. Targeting aptamers were then introduced to the particles through the reaction with the cross linker to target the nucleolin receptors over expressed on the cancer cell surface. Specific binding and uptake of the aptamer conjugated magnetic fluid loaded fluorescently tagged PLGA NPs (Apt-MF-NR-PLGA NPs) to the target cancer cells induced by aptamers was observed using confocal microscopy. Cytotoxicity assay conducted in two cell lines (L929 and MCF-7) confirmed that targeted MCF-7 cancer cells were killed while control cells were unharmed. In addition, aptamer mediated delivery resulting in enhanced binding and uptake to the target cancer cells exhibited increased therapeutic effect of the drug. Moreover, these aptamer conjugated magnetic polymer vehicles apart from actively transporting drugs into specifically targeted tumor regions can also be used to induce hyperthermia or for facilitating magnetic guiding of particles to the tumor regions. - Highlights: • Aptamer escorted, theranostic biodegradable PLGA carriers were developed. • Can target cancer cells, control drug release, image and magnetically guide. • Highly specific to the targeted cancer cells thus delivering

  16. Determination of cadmium at ultra-trace levels by CPE-molecular fluorescence combined methodology

    International Nuclear Information System (INIS)

    A highly sensitive micelle-mediated extraction methodology for the preconcentration and determination of trace levels of cadmium by molecular fluorescence has been developed. Metal was complexed with o-phenanthroline (o-phen) and eosin (eo) at pH 7.6 in buffer Tris medium and quantitatively extracted into a small volume of surfactant-rich phase of PONPE 7.5 after centrifugating. The chemical variables affecting cloud point extraction (CPE) were evaluated and optimized. The RSD for six replicates of cadmium determinations at 0.84 μg L-1 level was 1.17%. The linearity range using the preconcentration system was between 2.79 x 10-3 μg L-1 and 2.81 μg L-1 with a correlation coefficient of 0.99. Under the optimal conditions, it obtained a LOD of 8.38 x 10-4 μg L-1 and LOQ of 2.79 x 10-3 μg L-1. The method presented good sensitivity and selectivity and was applied to the determination of trace amounts of cadmium in commercially bottled mineral water, tap water and water well samples with satisfactory results. The proposed method is an innovative application of CPE-luminescence to metal analysis comparable in sensitivity and accuracy with atomic spectroscopies.

  17. Determination of cadmium at ultra-trace levels by CPE-molecular fluorescence combined methodology

    Energy Technology Data Exchange (ETDEWEB)

    Talio, Maria Carolina [Instituto de Quimica de San Luis (INQUISAL-CONICET), Chacabuco y Pedernera, 5700 San Luis (Argentina); Luconi, Marta O. [Area de Quimica Analitica, Facultad de Quimica, Bioquimica y Farmacia, Universidad Nacional de San Luis, San Luis (Argentina); Masi, Adriana N. [Area de Bromatologia- Ensayo y Valoracion de Medicamentos, Facultad de Quimica, Bioquimica y Farmacia, Universidad Nacional de San Luis, San Luis (Argentina); Instituto de Quimica de San Luis (INQUISAL-CONICET), Chacabuco y Pedernera, 5700 San Luis (Argentina); Fernandez, Liliana P., E-mail: lfernand@unsl.edu.ar [Area de Quimica Analitica, Facultad de Quimica, Bioquimica y Farmacia, Universidad Nacional de San Luis, San Luis (Argentina); Instituto de Quimica de San Luis (INQUISAL-CONICET), Chacabuco y Pedernera, 5700 San Luis (Argentina)

    2009-10-15

    A highly sensitive micelle-mediated extraction methodology for the preconcentration and determination of trace levels of cadmium by molecular fluorescence has been developed. Metal was complexed with o-phenanthroline (o-phen) and eosin (eo) at pH 7.6 in buffer Tris medium and quantitatively extracted into a small volume of surfactant-rich phase of PONPE 7.5 after centrifugating. The chemical variables affecting cloud point extraction (CPE) were evaluated and optimized. The RSD for six replicates of cadmium determinations at 0.84 {mu}g L{sup -1} level was 1.17%. The linearity range using the preconcentration system was between 2.79 x 10{sup -3} {mu}g L{sup -1} and 2.81 {mu}g L{sup -1} with a correlation coefficient of 0.99. Under the optimal conditions, it obtained a LOD of 8.38 x 10{sup -4} {mu}g L{sup -1} and LOQ of 2.79 x 10{sup -3} {mu}g L{sup -1}. The method presented good sensitivity and selectivity and was applied to the determination of trace amounts of cadmium in commercially bottled mineral water, tap water and water well samples with satisfactory results. The proposed method is an innovative application of CPE-luminescence to metal analysis comparable in sensitivity and accuracy with atomic spectroscopies.

  18. In Situ Live Cell Sensing of Multiple Nucleotides Exploiting DNA/RNA Aptamers and Graphene Oxide Nanosheets

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ying; Li, Zhaohui; Weber, Thomas J.; Hu, Dehong; Lin, Chiann Tso; Li, Jinghong; Lin, Yuehe

    2013-07-23

    Adenosine-5’-triphosphate (ATP) and guanosine-5’-triphosphate (GTP) are primary energy resources and function coordinately for numerous reactions such as microtubule assembly, insulin secretion and ion channel regulation. We have developed a novel DNA/RNA aptamer- graphene oxide nanosheet (GO-nS) sensing platform that can selectively and simultaneously detect ATP and GTP in live cells. A fluorescent tag is covalently attached to aptamers and fluorescence is quenched upon binding of aptamer to the GO-nS. Fluorescently tagged aptamers that selectively bind ATP or GTP were isolated from an aptamer library and were adsorbed onto GO-nS. Upon incubation with targets (ATP and/or GTP), the aptamers readily dissociated from GO-nS and the fluorescent signal was recovered. By covalently attaching fluorophores, both ATP and GTP sensing aptamers could be exploited to simultaneously visualize aptamer dissociation in live cells. In addition, the GO-nS appear to be biocompatible and protect the adsorbed DNA/RNA aptamers from enzymatic cleavage. Our results support the application of aptamer/GO-nS as a sensing platform for nucleotides in living cells and have implications for the development of additional sensor platforms for other bio-molecules that show selective interactions with aptamers and other biomarkers.

  19. Aptamer based electrochemical sensor system for protein using the generation/collection mode of scanning electrochemical microscope (SECM).

    Science.gov (United States)

    Park, Kyungsoon; Kwon, Dohyoung; Kwak, Juhyoun

    2011-05-01

    To detect the target molecules, aptamers are currently focused on and the use of aptamers for biosensing is particularly interesting, as aptamers could substitute antibodies in bioanalytical sensing. So this paper describes the novel electrochemical system for protein in sandwich manner by using the aptamers and the scanning electrochemical microscope (SECM). For protein detection, sandwich system is ideal since labeling of the target protein is not necessary. To develop the electrochemical protein sensor system, thrombin was chosen as a target protein since many aptamers for it were already reported and two different aptamers, which recognize different positions of thrombin, were chosen to construct sandwich type sensing system. In order to obtain the electrochemical signal, the glucose oxidase (GOD) used for labeling the detection aptamers since it has large amount of stability in aqueous solution. One aptamer was immobilized onto the gold electrode and the other aptamer for detection was labeled with GOD for generation of the electric signal. Thrombin was detected in sandwich manner with aptamer immobilized onto the gold electrode and the GOD labeled aptamer. The enzymatic signal, generated from glucose addition after the formation of the complex of thrombin, was measured. The generation-collection mode of SECM was used for amperometric H2O2 detection. PMID:21780447

  20. Characterisation of aptamer-target interactions by branched selection and high-throughput sequencing of SELEX pools

    DEFF Research Database (Denmark)

    Dupont, Daniel M; Larsen, Niels; Jensen, Jan K;

    2015-01-01

    Nucleic acid aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX) has shown great promise for use in the development of research tools, therapeutics and diagnostics. Typically, aptamers are identified from libraries containing up to 10(16) different RNA or DNA...... sequences by 5-10 rounds of affinity selection towards a target of interest. Such library screenings can result in complex pools of many target-binding aptamers. New high-throughput sequencing techniques may potentially revolutionise aptamer selection by allowing quantitative assessment of the dynamic...... provide detailed information about aptamer binding sites, preferences for specific target conformations, and functional effects of the aptamers. The procedure was applied on a diverse pool of 2'-fluoropyrimidine-modified RNA enriched for aptamers specific for the serpin plasminogen activator inhibitor-1...

  1. Solid-contact potentiometric aptasensor based on aptamer functionalized carbon nanotubes for the direct determination of proteins.

    Science.gov (United States)

    Düzgün, Ali; Maroto, Alicia; Mairal, Teresa; O'Sullivan, Ciara; Rius, F Xavier

    2010-05-01

    A facile, solid-contact selective potentiometric aptasensor exploiting a network of single-walled carbon nanotubes (SWCNT) acting as a transducing element is described in this work. The molecular properties of the SWCNT surface have been modified by covalently linking aptamers as biorecognition elements to the carboxylic groups of the SWCNT walls. As a model system to demonstrate the generic application of the approach, a 15-mer thrombin aptamer interacts with thrombin and the affinity interaction gives rise to a direct potentiometric signal that can be easily recorded within 15 s. The dynamic linear range, with a sensitivity of 8.0 mV/log a(Thr) corresponds to the 10(-7)-10(-6) M range of thrombin concentrations, with a limit of detection of 80 nM. The aptasensor displays selectivity against elastase and bovine serum albumin and is easily regenerated by immersion in 2 M NaCl. The aptasensor demonstrates the capacity of direct detection of the recognition event avoiding the use of labels, mediators, or the addition of further reagents or analyte accumulation. PMID:20419254

  2. Rational design of a redox-labeled chiral target for an enantioselective aptamer-based electrochemical binding assay.

    Science.gov (United States)

    Moreau, Julie; Challier, Lylian; Lalaoui, Noémie; Mavré, François; Noël, Vincent; Limoges, Benoît; Schöllhorn, Bernd; Fave, Claire

    2014-03-01

    A series of redox-labeled L-tyrosinamide (L-Tym) derivatives was prepared and the nature of the functional group and the chain length of the spacer were systematically varied in a step-by-step affinity optimization process of the tracer for the L-Tym aptamer. The choice of the labeling position on L-Tym proved to be crucial for the molecular recognition event, which could be monitored by cyclic voltammetry and is based on the different diffusion rates of free and bound targets in solution. From this screening approach an efficient electroactive tracer emerged. Comparable dissociation constants Kd were obtained for the unlabeled and labeled targets in direct or competitive binding assays. The enantiomeric tracer was prepared and its enantioselective recognition by the corresponding anti-D-Tym aptamer was demonstrated. The access to both enantiomeric tracer molecules opens the door for the development of one-pot determination of the enantiomeric excess when using different labels with well-separated redox potentials for each enantiomer. PMID:24519626

  3. The G-quadruplex-forming aptamer AS1411 potently inhibits HIV-1 attachment to the host cell.

    Science.gov (United States)

    Perrone, Rosalba; Butovskaya, Elena; Lago, Sara; Garzino-Demo, Alfredo; Pannecouque, Christophe; Palù, Giorgio; Richter, Sara N

    2016-04-01

    AS1411 is a G-rich aptamer that forms a stable G-quadruplex structure and displays antineoplastic properties both in vitro and in vivo. This oligonucleotide has undergone phase 2 clinical trials. The major molecular target of AS1411 is nucleolin (NCL), a multifunctional nucleolar protein also present in the cell membrane where it selectively mediates the binding and uptake of AS1411. Cell-surface NCL has been recognised as a low-affinity co-receptor for human immunodeficiency virus type 1 (HIV-1) anchorage on target cells. Here we assessed the anti-HIV-1 properties and underlying mechanism of action of AS1411. The antiviral activity of AS1411 was determined towards different HIV-1 strains, host cells and at various times post-infection. Acutely, persistently and latently infected cells were tested, including HIV-1-infected peripheral blood mononuclear cells from a healthy donor. Mechanistic studies to exclude modes of action other than virus binding via NCL were performed. AS1411 efficiently inhibited HIV-1 attachment/entry into the host cell. The aptamer displayed antiviral activity in the absence of cytotoxicity at the tested doses, therefore displaying a wide therapeutic window and favourable selectivity indexes. These findings, besides validating cell-surface-expressed NCL as an antiviral target, open the way for the possible use of AS1411 as a new potent and promisingly safe anti-HIV-1 agent. PMID:27032748

  4. The G-quadruplex-forming aptamer AS1411 potently inhibits HIV-1 attachment to the host cell

    Science.gov (United States)

    Perrone, Rosalba; Butovskaya, Elena; Lago, Sara; Garzino-Demo, Alfredo; Pannecouque, Christophe; Palù, Giorgio; Richter, Sara N.

    2016-01-01

    AS1411 is a G-rich aptamer that forms a stable G-quadruplex structure and displays antineoplastic properties both in vitro and in vivo. This oligonucleotide has undergone phase 2 clinical trials. The major molecular target of AS1411 is nucleolin (NCL), a multifunctional nucleolar protein also present in the cell membrane where it selectively mediates the binding and uptake of AS1411. Cell-surface NCL has been recognised as a low-affinity co-receptor for human immunodeficiency virus type 1 (HIV-1) anchorage on target cells. Here we assessed the anti-HIV-1 properties and underlying mechanism of action of AS1411. The antiviral activity of AS1411 was determined towards different HIV-1 strains, host cells and at various times post-infection. Acutely, persistently and latently infected cells were tested, including HIV-1-infected peripheral blood mononuclear cells from a healthy donor. Mechanistic studies to exclude modes of action other than virus binding via NCL were performed. AS1411 efficiently inhibited HIV-1 attachment/entry into the host cell. The aptamer displayed antiviral activity in the absence of cytotoxicity at the tested doses, therefore displaying a wide therapeutic window and favourable selectivity indexes. These findings, besides validating cell-surface-expressed NCL as an antiviral target, open the way for the possible use of AS1411 as a new potent and promisingly safe anti-HIV-1 agent. PMID:27032748

  5. Studies of DNA Aptamer OliGreen and PicoGreen Fluorescence Interactions in Buffer and Serum.

    Science.gov (United States)

    Bruno, John G; Sivils, Jeffrey C

    2016-07-01

    Spectrofluorometric and emission peak titration and timed studies of OliGreen (OG) and PicoGreen (PG) were conducted in Tris EDTA (TE) buffer, pooled rat and fetal bovine serum with two different aptamers of 72 and 192 bases in length to determine if OG or PG were suitable for aptamer pharmacokinetic (PK) studies in sera. Results indicated that OG and PG detected the single-stranded (ss) and double-stranded (ds) stem-loop structures of the two aptamers quite well in TE with reliable standard curves having exponential character (or several linear detection regions) up to 1 μg/ml of aptamer DNA with detection limits of ~1 ng/ml. The intensity of OG and PG staining appeared to correlate with the number and percentage of ss and ds bases in each aptamer. OG and PG fluorescence in pooled rat serum or fetal bovine serum (FBS) did not titer as a function of DNA aptamer concentration from 1 μg/ml to 1 ng/ml. This lack of OG or PG aptamer assays in serum is contrary to most published reports of OG or PG assays for ss antisense oligonucleotides, ds PCR amplicons or other types of DNA in serum or plasma. Further studies suggested that the lack of OG and PG assay titration in serum might not be entirely due to aptamer degradation from nucleases in serum since the fluorescence signals in serum appeared relatively stable over time from 30 min to 4 hours. A hypothesis is presented which attributes the inability of OG or PG to assay aptamers in serum to a combination of high blue-green autofluorescence in serum with possible serum nuclease degradation of aptamers over time and the changing aptamer to serum protein ratio coupled to nonspecific binding of serum proteins to aptamers thereby possibly changing aptamer conformations as a function of aptamer concentration during titration experiments. PMID:27209004

  6. Development, screening, and analysis of DNA aptamer libraries potentially useful for diagnosis and passive immunity of arboviruses

    Directory of Open Access Journals (Sweden)

    Bruno John G

    2012-11-01

    Full Text Available Abstract Background Nucleic acid aptamers have long demonstrated the capacity to bind viral envelope proteins and to inhibit the progression of pathogenic virus infections. Here we report on initial efforts to develop and screen DNA aptamers against recombinant envelope proteins or synthetic peptides and whole inactivated viruses from several virulent arboviruses including Chikungunya, Crimean-Congo hemorrhagic fever (CCHF, dengue, tickborne encephalitis and West Nile viruses. We also analyzed sequence data and secondary structures for commonalities that might reveal consensus binding sites among the various aptamers. Some of the highest affinity and most specific aptamers in the down-selected libraries were demonstrated to have diagnostic utility in lateral flow chromatographic assays and in a fluorescent aptamer-magnetic bead sandwich assay. Some of the reported aptamers may also be able to bind viral envelope proteins in vivo and therefore may have antiviral potential in passive immunity or prophylactic applications. Results Several arbovirus DNA aptamer sequences emerged multiple times in the various down selected aptamer libraries thereby suggesting some consensus sequences for binding arbovirus envelope proteins. Screening of aptamers by enzyme-linked aptamer sorbent assay (ELASA was useful for ranking relative aptamer affinities against their cognate viral targets. Additional study of the aptamer sequences and secondary structures of top-ranked anti-arboviral aptamers suggest potential virus binding motifs exist within some of the key aptamers and are highlighted in the supplemental figures for this article. One sequence segment (ACGGGTCCGGACA emerged 60 times in the anti-CCHF aptamer library, but nowhere else in the anti-arbovirus library and only a few other times in a larger library of aptamers known to bind bacteria and rickettsia or other targets. Diagnostic utility of some of the aptamers for arbovirus detection in lateral flow

  7. Anthropogenic and Climate Influences on Biogeochemical Dynamics and Molecular-Level Speciation of Soil Sulfur

    Energy Technology Data Exchange (ETDEWEB)

    Solomon, D.; Lehmann, J; Kinyangi, J; Pell, A; Theis , J; Riha , S; Ngoze, S; Amelung, W; du Preez, C; et. al.

    2009-01-01

    The soil environment is a primary component of the global biogeochemical sulfur (S) cycle, acting as a source and sink of various S species and mediating oxidation state changes. However, ecological significance of the various S forms and the impacts of human intervention and climate on the amount and structural composition of these compounds are still poorly understood. We investigated the long-term influences of anthropogenically mediated transitions from natural to managed ecosystems on molecular-level speciation, biogeochemical dynamics, and the apparent temperature sensitivity of S moieties in temperate, subtropical, and tropical environments with mean annual temperature (MAT) ranging from 5C to 21C, using elemental analysis and X-ray absorption near-edge structure (XANES) spectroscopy. Land-use and land-cover changes led to the depletion of total soil S in all three ecoregions over a period of up to 103 years. The largest decline occurred from tropical forest agroecosystems (67% Kakamega and 76% Nandi, Kenya), compared to losses from temperate (36% at Lethbridge, Canada, and 40% at Pendleton, USA) and subtropical (48% at South Africa) grassland agroecosystems. The total S losses correlated significantly with MAT. Anthropogenic interventions profoundly altered the molecular-level composition and resulted in an apparent shift in oxidation states of organic S from native ecosystems composed primarily of S moieties in intermediate and highly reduced oxidation states toward managed agroecosystems dominated by organic S rich in strongly oxidized functionalities. The most prominent change occurred in thiols and sulfides, the proportion of which decreased by 46% (Lethbridge) and 57% (Pendleton) in temperate agroecosystems, by 46% in subtropical agroecosystems, and by 79% (Nandi) and 81% (Kakamega) in tropical agroecosystems. The proportion of organic S directly linked to O increased by 81%, 168%, 40%, 92%, and 85%, respectively. Among the various organic S

  8. Two-level hierarchical fragmentation in the northern filament of the Orion Molecular Cloud 1

    Science.gov (United States)

    Teixeira, P. S.; Takahashi, S.; Zapata, L. A.; Ho, P. T. P.

    2016-03-01

    Context. The filamentary structure of molecular clouds may set important constraints on the mass distribution of stars forming within them. It is therefore important to understand which physical mechanism dominates filamentary cloud fragmentation and core formation. Aims: Orion A is the nearest giant molecular cloud, and its so-called ∫-shaped filament is a very active star-forming region that is a good target for such a study. We have recently reported on the collapse and fragmentation properties of the northernmost part of this structure, located ~2.4 pc north of Orion KL - Orion Molecular Cloud (OMC) 3. As part of our project to study the ∫-shaped filament, we analyze the fragmentation properties of the northern OMC 1 filament (located ≲0.3 pc north of Orion KL). This filament is a dense structure previously identified by JCMT/SCUBA submillimeter continuum and VLA NH3 observations and was shown to have fragmented into clumps. Our aim is to search for cores and young protostars embedded within OMC 1n and to study how the filament is fragmenting to form them. Methods: We observed OMC 1North (hereafter OMC 1n) with the Submillimeter Array (SMA) at 1.3 mm and report on our analysis of the continuum data. Results: We discovered 24 new compact sources, ranging in mass from 0.1 to 2.3, in size from 400 to 1300 au, and in density from 2.6 × 107 to 2.8 × 106 cm-3. The masses of these sources are similar to those of the SMA protostars in OMC 3, but their typical sizes and densities are lower by a factor of ten. Only 8% of the new sources have infrared counterparts, but there are five associated CO molecular outflows. These sources are thus likely in the Class 0 evolutionary phase but it cannot be excluded that some of the sources might still be pre-stellar cores. The spatial analysis of the protostars shows that they are divided into small groups that coincide with previously identified JCMT/SCUBA 850 μm and VLA NH3 clumps, which are separated by a quasi

  9. Polarized emission from stretched PPV films viewed at the molecular level.

    Science.gov (United States)

    Ramos, Rodrigo; Siqueira, Melissa F; Cazati, Thiago; Faria, Roberto M; Caldas, Marilia J

    2015-08-28

    We present a study on the photoluminescence (PL) of thin films of poly-(p-phenylene vinylene) (PPV), non-stretched and uniaxially stretched. The experimental study was carried out using linear polarized light as the excitation beam, oriented either parallel or perpendicular to the stretch axis (S). The results showed that when the excitation light source has polarization perpendicularly oriented to the stretch direction, the emitted PL presents maximum intensity in the orientation S, and a minimum in the direction orthogonal to S. In order to understand this interesting phenomenon, we employ theoretical simulations at the atomistic level. We use classical molecular dynamics to simulate amorphous PPV films, non-stretched and stretched, from which we find a tendency of overall alignment of PV units to S, and of local clustering in herring-bone and π-stacking partial symmetries. Our study of optical activity of these kinds of clusters, performed through a quantum semi-empirical method, allows us to explain this polarization conversion behavior, and indicates the possibility of using underivatized PPV as the active layer for polarized electroluminescent devices. PMID:26198411

  10. Insights into surface–adsorbate interactions in corrosion inhibition processes at the molecular level

    International Nuclear Information System (INIS)

    Graphical abstract: The interaction of 2-((3-methylpyridine-2-imino)methyl)phenol (MPIMP) with the Fe(1 1 0) surface was clarified at the molecular level using density functional theory (DFT). Highlights: •2-((3-Methylpyridine-2-imino)methyl)phenol was tested as a corrosion inhibitor. •Its interaction with the surface was characterized using density functional theory. •Three stable adsorption configurations on Fe(1 1 0) surface were identified. -- Abstract: 2-((3-Methylpyridine-2-imino)methyl)phenol (MPIMP) was investigated as a potential corrosion inhibitor for mild steel in 0.5 M HCl solution using impedance spectroscopy (IS). Changes in impedance parameters indicated that adsorption of MPIMP occurred on the mild steel surface. Three stable adsorption configurations for MPIMP on the Fe(1 1 0) surface were identified as a result of geometry optimization starting from several adsorption geometries using density functional theory (DFT). Involvement of the delocalized π-electrons of the aromatic rings in the interaction provides extra stabilization to the flat adsorption configurations

  11. Energy level alignment and electron transport through metal/organic contacts. From interfaces to molecular electronics

    Energy Technology Data Exchange (ETDEWEB)

    Abad, Enrique

    2013-07-01

    A new calculational approach to describing metal/organic interfaces. A valuable step towards a better understanding of molecular electronics. Nominated as an outstanding contribution by the Autonomous University of Madrid. In recent years, ever more electronic devices have started to exploit the advantages of organic semiconductors. The work reported in this thesis focuses on analyzing theoretically the energy level alignment of different metal/organic interfaces, necessary to tailor devices with good performance. Traditional methods based on density functional theory (DFT), are not appropriate for analyzing them because they underestimate the organic energy gap and fail to correctly describe the van der Waals forces. Since the size of these systems prohibits the use of more accurate methods, corrections to those DFT drawbacks are desirable. In this work a combination of a standard DFT calculation with the inclusion of the charging energy (U) of the molecule, calculated from first principles, is presented. Regarding the dispersion forces, incorrect long range interaction is substituted by a van der Waals potential. With these corrections, the C60, benzene, pentacene, TTF and TCNQ/Au(111) interfaces are analyzed, both for single molecules and for a monolayer. The results validate the induced density of interface states model.

  12. Assessment of radiation-induced damage to DNA at the molecular level by mass spectrometry

    International Nuclear Information System (INIS)

    Full text: Ionizing radiation-induced damage to DNA in living cells may lead to biological consequences such as mutation, carcinogenesis or cell death. Free radicals generated from water by ionising radiation, especially OH radicals produce a large number of base and sugar derived stable products in DNA as well as DNA-protein complexes. Repair of these structural modifications is essential to maintain the genomic integrity to prevent long term effects of radiation. Understanding the cellular repair of ionizing radiation-induced damage to DNA in cell obviously depends upon the knowledge of the chemical nature of DNA lesions and their quantities at the molecular level. Several types of assays are known to measure the yields of radiation-induced purine and pyrimidine products in DNA which include chromatographic, immunochemical or biochemical technologies. However, these procedures are capable of assaying only a very limited number of modified DNA products and lack sensitivity and selectivity. We have developed sensitive and specific gas chromatography-mass spectrometry methods (GC/MS) for chemical identification and estimation of modifications in DNA at physiologically relevant doses. This presentation will focus primarily on the sampling, instrumentation, applications and limitations of GC/MS procedures in assaying radiation-induced damage and repair to DNA

  13. Nucleic-Acid-Binding Chromophores as Efficient Indicators of Aptamer-Target Interactions

    Directory of Open Access Journals (Sweden)

    Kwabena Sarpong

    2012-01-01

    Full Text Available The binding affinity and specificity of nucleic acid aptamers have made them valuable candidates for use as sensors in diagnostic applications. In particular, chromophore-functionalized aptamers offer a relatively simple format for detection and quantification of target molecules. We describe the use of nucleic-acid-staining reagents as an effective tool for detecting and signaling aptamer-target interactions. Aptamers varying in size and structure and targeting a range of molecules have been used in conjunction with commercially available chromophores to indicate and quantify the presence of cognate targets with high sensitivity and selectivity. Our assay precludes the covalent modification of nucleic acids and relies on the differential fluorescence signal of chromophores when complexed with aptamers with or without their cognate target. We also evaluate factors that are critical for the stability of the complex between the aptamer and chromophore in presence or absence of target molecules. Our results indicate the possibility of controlling those factors to enhance the sensitivity of target detection by the aptamers used in such assays.

  14. 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.

  15. 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.

  16. 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.

  17. Surface biofunctionalization of β-TCP blocks using aptamer 74 for bone tissue engineering.

    Science.gov (United States)

    Ardjomandi, N; Huth, J; Stamov, D R; Henrich, A; Klein, C; Wendel, H-P; Reinert, S; Alexander, D

    2016-10-01

    Successful bone regeneration following oral and maxillofacial surgeries depends on efficient functionalization strategies that allow the recruitment of osteogenic progenitor cells at the tissue/implant interface. We have previously identified aptamer 74, which exhibited a binding affinity for osteogenically induced jaw periosteal cells (JPCs). In the present study, this aptamer was used for the surface biofunctionalization of β-tricalcium phosphate (β-TCP) blocks. Atomic force microscopy (AFM) measurements showed increased binding activity of aptamer 74 towards osteogenically induced JPCs compared to untreated controls. The immobilization efficiency of aptamer 74 was analyzed using the QuantiFluor ssDNA assay for 2D surfaces and by amino acid analysis for 3D β-TCP constructs. Following the successful immobilization of aptamer 74 in 2D culture wells and on 3D constructs, in vitro assays showed no significant differences in cell proliferation compared to unmodified surfaces. Interestingly, JPC mineralization was significantly higher on the 2D surfaces and higher cell adhesion was detected on the 3D constructs with immobilized aptamer. Herein, we report an established, biocompatible β-TCP matrix with surface immobilization of aptamer 74, which enhances properties such as cell adhesion on 3D constructs and mineralization on 2D surfaces. Further studies need to be performed to improve the immobilization efficiency and to develop a suitable approach for JPC mineralization growing within 3D β-TCP constructs. PMID:27287122

  18. Enzymatic conjugation of multiple proteins on a DNA aptamer in a tail-specific manner.

    Science.gov (United States)

    Takahara, Mari; Hayashi, Kounosuke; Goto, Masahiro; Kamiya, Noriho

    2016-06-01

    Conjugation of single-strand DNA aptamers and enzymes has been of great significance in bioanalytical and biomedical applications because of the unlimited functions provided by DNA aptamer direction. Therefore, we developed efficient tailing of a DNA aptamer, with end-specific conjugation of multiple enzymes, through enzymatic catalysis. Terminal deoxynucleotidyl transferase (TdT) added multiple Z-Gln-Gly (Z-QG) moieties to the 3'-end of a DNA aptamer via the addition of Z-QG-modified deoxyuridine triphosphate (Z-QG-dUTP) and deoxynucleoside triphosphates (dNTPs). The resultant (Z-QG)m -(dN)l-aptamer, whose Z-QGs with dN spacers served as stickers for microbial transglutaminase (MTG), were crosslinked between the Z-QGs on the DNA and a substrate peptide sequence containing lysine (K), fused to a recombinant enzyme (i.e. bacterial alkaline phosphatase; BAP) by MTG. The incorporation efficiency of Z-QG moieties on the aptamer tail and the subsequent conjugation efficiency with multiple enzyme molecules were dramatically altered by the presence of dNTPs, revealing that a combination of Z-QG-dUTP/dTTP comprised the best labeling efficiency and corresponding properties during analytical performance. Thus, a novel optimized platform for designing (BAP)n -(dT)l-DNA aptamers was demonstrated for the first time in this article, offering unique opportunities for tailoring new types of covalent protein-nucleic acid conjugates in a controllable way. PMID:27119459

  19. Dual-colour imaging of RNAs using quencher- and fluorophore-binding aptamers.

    Science.gov (United States)

    Arora, Ankita; Sunbul, Murat; Jäschke, Andres

    2015-12-01

    In order to gain deeper insight into the functions and dynamics of RNA in cells, the development of methods for imaging multiple RNAs simultaneously is of paramount importance. Here, we describe a modular approach to image RNA in living cells using an RNA aptamer that binds to dinitroaniline, an efficient general contact quencher. Dinitroaniline quenches the fluorescence of different fluorophores when directly conjugated to them via ethylene glycol linkers by forming a non-fluorescent intramolecular complex. Since the binding of the RNA aptamer to the quencher destroys the fluorophore-quencher complex, fluorescence increases dramatically upon binding. Using this principle, a series of fluorophores were turned into fluorescent turn-on probes by conjugating them to dinitroaniline. These probes ranged from fluorescein-dinitroaniline (green) to TexasRed-dinitroaniline (red) spanning across the visible spectrum. The dinitroaniline-binding aptamer (DNB) was generated by in vitro selection, and was found to bind all probes, leading to fluorescence increase in vitro and in living cells. When expressed in E. coli, the DNB aptamer could be labelled and visualized with different-coloured fluorophores and therefore it can be used as a genetically encoded tag to image target RNAs. Furthermore, combining contact-quenched fluorogenic probes with orthogonal DNB (the quencher-binding RNA aptamer) and SRB-2 aptamers (a fluorophore-binding RNA aptamer) allowed dual-colour imaging of two different fluorescence-enhancing RNA tags in living cells, opening new avenues for studying RNA co-localization and trafficking. PMID:26175046

  20. Graphene-based aptamer logic gates and their application to multiplex detection.

    Science.gov (United States)

    Wang, Li; Zhu, Jinbo; Han, Lei; Jin, Lihua; Zhu, Chengzhou; Wang, Erkang; Dong, Shaojun

    2012-08-28

    In this work, a GO/aptamer system was constructed to create multiplex logic operations and enable sensing of multiplex targets. 6-Carboxyfluorescein (FAM)-labeled adenosine triphosphate binding aptamer (ABA) and FAM-labeled thrombin binding aptamer (TBA) were first adsorbed onto graphene oxide (GO) to form a GO/aptamer complex, leading to the quenching of the fluorescence of FAM. We demonstrated that the unique GO/aptamer interaction and the specific aptamer-target recognition in the target/GO/aptamer system were programmable and could be utilized to regulate the fluorescence of FAM via OR and INHIBIT logic gates. The fluorescence changed according to different input combinations, and the integration of OR and INHIBIT logic gates provided an interesting approach for logic sensing applications where multiple target molecules were present. High-throughput fluorescence imagings that enabled the simultaneous processing of many samples by using the combinatorial logic gates were realized. The developed logic gates may find applications in further development of DNA circuits and advanced sensors for the identification of multiple targets in complex chemical environments. PMID:22823159

  1. Imaging gene expression in real-time using aptamers

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Ilchung [Iowa State Univ., Ames, IA (United States)

    2012-01-01

    Signal transduction pathways are usually activated by external stimuli and are transient. The downstream changes such as transcription of the activated genes are also transient. Real-time detection of promoter activity is useful for understanding changes in gene expression, especially during cell differentiation and in development. A simple and reliable method for viewing gene expression in real time is not yet available. Reporter proteins such as fluorescent proteins and luciferase allow for non-invasive detection of the products of gene expression in living cells. However, current reporter systems do not provide for real-time imaging of promoter activity in living cells. This is because of the long time period after transcription required for fluorescent protein synthesis and maturation. We have developed an RNA reporter system for imaging in real-time to detect changes in promoter activity as they occur. The RNA reporter uses strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags), which can be expressed from a promoter of choice. The tobramycin, neomycin and PDC RNA aptamers have been utilized for this system and expressed in yeast from the GAL1 promoter. The IMAGEtag RNA kinetics were quantified by RT-qPCR. In yeast precultured in raffinose containing media the GAL1 promoter responded faster than in yeast precultured in glucose containing media. IMAGEtag RNA has relatively short half-life (5.5 min) in yeast. For imaging, the yeast cells are incubated with their ligands that are labeled with fluorescent dyes. To increase signal to noise, ligands have been separately conjugated with the FRET (Förster resonance energy transfer) pairs, Cy3 and Cy5. With these constructs, the transcribed aptamers can be imaged after activation of the promoter by galactose. FRET was confirmed with three different approaches, which were sensitized emission, acceptor photobleaching and donor lifetime by FLIM (fluorescence lifetime imaging

  2. Imaging gene expression in real-time using aptamers

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Il Chung [Iowa State Univ., Ames, IA (United States)

    2011-01-01

    Signal transduction pathways are usually activated by external stimuli and are transient. The downstream changes such as transcription of the activated genes are also transient. Real-time detection of promoter activity is useful for understanding changes in gene expression, especially during cell differentiation and in development. A simple and reliable method for viewing gene expression in real time is not yet available. Reporter proteins such as fluorescent proteins and luciferase allow for non-invasive detection of the products of gene expression in living cells. However, current reporter systems do not provide for real-time imaging of promoter activity in living cells. This is because of the long time period after transcription required for fluorescent protein synthesis and maturation. We have developed an RNA reporter system for imaging in real-time to detect changes in promoter activity as they occur. The RNA reporter uses strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags), which can be expressed from a promoter of choice. The tobramycin, neomycin and PDC RNA aptamers have been utilized for this system and expressed in yeast from the GAL1 promoter. The IMAGEtag RNA kinetics were quantified by RT-qPCR. In yeast precultured in raffinose containing media the GAL1 promoter responded faster than in yeast precultured in glucose containing media. IMAGEtag RNA has relatively short half-life (5.5 min) in yeast. For imaging, the yeast cells are incubated with their ligands that are labeled with fluorescent dyes. To increase signal to noise, ligands have been separately conjugated with the FRET (Förster resonance energy transfer) pairs, Cy3 and Cy5. With these constructs, the transcribed aptamers can be imaged after activation of the promoter by galactose. FRET was confirmed with three different approaches, which were sensitized emission, acceptor photobleaching and donor lifetime by FLIM (fluorescence lifetime imaging

  3. Antibody- and aptamer-strategies for GvHD prevention.

    Science.gov (United States)

    Oelkrug, Christopher; Sack, Ulrich; Boldt, Andreas; Nascimento, Isis C; Ulrich, Henning; Fricke, Stephan

    2015-01-01

    Prevention of Graft-versus-Host-Disease (GvHD) by preserved Graft-versus-Leukaemia (GvL) effect is one of the major obstacles following allogeneic haematopoietic stem cell transplantation. Currently used drugs are associated with side effects and were not able to separate GvHD from the GvL-effect because of general T-cell suppression. This review focuses on murine models for GvHD and currently available treatment options involving antibodies and applications for the therapeutic use of aptamers as well as strategies for targeting immune responses by allogenic antigens. PMID:25353670

  4. Surface plasmon resonance imaging (SPRi) for analysis of DNA aptamer:β-conglutin interactions.

    Science.gov (United States)

    Jauset Rubio, Miriam; Svobodová, Markéta; Mairal, Teresa; O'Sullivan, Ciara K

    2016-03-15

    Surface plasmon resonance imaging (SPRi) is a label-free detection method that offers a suitable and reliable platform for the real time monitoring of biomolecular interactions. In the work reported here, SPRi was used to evaluate the affinity and specificity of three different aptamers selected against the Lup an 1 anaphylactic allergen β-conglutin (β-conglutin binding aptamers I and II (β-CBA I and β-CBA II)), as well as an 11-mer truncated version of β-CBA I. Thiol modified aptamers were immobilised on a gold substrate through a self-assembling process and the use of different blocking strategies to prevent non-specific binding were evaluated. Dissociation constants of 20, 13 and 1 nM were determined for β-CBA I, β-CBA II and the 11-mer truncated aptamer, respectively. The three aptamers were then studied in various different sandwich formats and the β-CBA I/11-mer and β-CBA II were observed to bind to different aptatopes on the target protein. Each of the aptamers were then used either as surface immobilised aptamer, or as reporter aptamer, and added with the protein target β-conglutin in either a sequential of simultaneous manner, and the changes in SPR signal monitored. The preferred approach for formation of a sandwich aptacomplex was with immobilised β-CBA II, followed by addition of pre-incubated β-conglutin and 11-mer, whilst addition of the 11-mer following addition of the β-conglutin, resulted in displacement of the bound target. The ability to provide parallel qualitative and quantitative detection establishes SPRi as a powerful tool for the study of immobilised aptamer-target interactions. PMID:26515644

  5. 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. PMID:26768582

  6. Robust nanoplasmonic substrates for aptamer macroarrays with single-step detection of PDGF-BB.

    Science.gov (United States)

    Zhu, Dong; Yang, Rui Xiang; Tang, Yu-Ping; Li, Wei; Miao, Zhao Yi; Hu, Yue; Chen, Jun; Yu, Sheng; Wang, Juan; Xu, Chen Yang

    2016-11-15

    An aptamer macroarray on a robust nanoplasmonic substrate with fluorescence enhancement is developed for a single-step sensitive detection of human platelet-derived growth factor-BB (PDGF-BB), a predominant cancer biomarker in cancer angiogenesis. A hybrid Au-nanoparticles-poly (dimethylsiloxane) (PDMS) as nanoplasmonic substrate is prepared via the in-situ reduction of AuCl4(-) ions in PDMS matrixes onto 96 or 384 well plates. In the absence of target molecules, unfolded PDGF-BB aptamer conjugated with dye TAMRA is electrostatically bound to a positively charged poly-L-lysine (PLL)-coated Au nanocomposites film surface, and the fluorescence enhancement effects can be optimized by varying the distance between TAMRA and the Au nanocomposites film, which is easily adjusted by varying the thickness of the biocompatible poly-(acrylic acid) (PAA/PLL) multilayers, and thus metal-enhanced fluorescence of dye TAMRA conjugated with the aptamer is generated up to 15.2-fold. The interaction of the aptamer to its target induces the reversible conformation change of the aptamer, and consequently, the electrostatic potential is overcome by binding force. As a result, the target-binding interaction of the aptamer causes the irreversible detachment of the aptamer from the nanostructured Au film surface to decrease fluorescence of TAMRA. The aptamer macroarray provides not only the appropriate sensitivity for clinical diagnostics with a wide range of linear detection from 10pg/mL to 10μg/mL, high specificity for PDGF-BB against VEGF-165, VEGF-121, NaCl and IgG, and temporal biological stability, but also a single-step detection. We envision that the efficient and robust aptamer macroarray can be extended to the detection of other biomarkers. PMID:27208474

  7. In situ sensing and modeling of molecular events at the cellular level

    Science.gov (United States)

    Yang, Ruiguo

    We developed the Atomic Force Microscopy (AFM) based nanorobot in combination with other nanomechanical sensors for the investigation of cell signaling pathways. The AFM nanorobotics hinge on the superior spatial resolution of AFM in imaging and extends it into the measurement of biological processes and manipulation of biological matters. A multiple input single output control system was designed and implemented to solve the issues of nanomanipulation of biological materials, feedback, response frequency and nonlinearity. The AFM nanorobotic system therefore provide the human-directed position, velocity and force control with high frequency feedback, and more importantly it can feed the operator with the real-time imaging of manipulation result from the fast-imaging based local scanning. The use of the system has taken the study of cellular process at the molecular scale into a new level. The cellular response to the physiological conditions can be significantly manifested in cellular mechanics. Dynamic mechanical property has been regarded as biomarkers, sometimes even regulators of the signaling and physiological processes, thus the name mechanobiology. We sought to characterize the relationship between the structural dynamics and the molecular dynamics and the role of them in the regulation of cell behavior. We used the AFM nanorobotics to investigate the mechanical properties in real-time of cells that are stimulated by different chemical species. These reagents could result in similar ion channel responses but distinctive mechanical behaviors. We applied these measurement results to establish a model that describes the cellular stimulation and the mechanical property change, a "two-hit" model that comprises the loss of cell adhesion and the initiation of cell apoptosis. The first hit was verified by functional experiments: depletion of Calcium and nanosurgery to disrupt the cellular adhesion. The second hit was tested by a labeling of apoptotic markers that

  8. Heat impact caused molecular level changes in solid and dissolved soil organic matter

    Science.gov (United States)

    Hofmann, Diana; Steffen, Bernhard; Eckhardt, Kai-Uwe; Leinweber, Peter

    2015-04-01

    resolution (used 400.000 at m/z 400 Da) and mass accuracy (≤ 1 ppm), simultaneously providing molecular level details of thousands of compounds. The characteristics and differences of the FTICR-MS spectra with as many as ten or more peaks at each nominal mass are discussed: heated samples showed considerable higher intensities of even numbered peaks. An in-house developed, automated post processing was used for further exploitation of the data with the aim of an unambiguous assignment of as many peaks as possible. Obtained mass lists were transformed for sorting and preparation/ interpretation of graphics like Kendrick and van Krevelen plots. The heat-treated solid samples show decreasing C/N ratios and the formation cyclic and N-heterocyclic compounds in good agreement among the various methods (Py-FIMS and C- and N-XANES). Detailed insight into the hot-water extracts by FTICR-MS showed clear qualitative as well as quantitative changes in the number and the intensity of nitrogen and nitrogen + sulfur containing compounds, respectively, which generally became enriched under soil heating. This demonstrates for the first time, that not only the bulk SOM is affected in structure by heat impact but also the more mobile DOM. We assume, that heat impact volatilizes and oxidizes parts of the organic substances is as expected but another part of the substances incorporates (further) nitrogen atom(s) similar to the generation of new compounds under the conditions of plasma etching in nitrogen atmosphere. This would explain to some extent, why soils are e.g. after fire clearing of vegetation are highly fertile for a short period (better plant acceptable compounds) but become more infertile in the long run, especially under tropical conditions with frequently heavy rain that would lead to an increased leaching of compounds with higher polarity.

  9. Aptamers and methods for their in vitro selection and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Doyle, Sharon A. (Walnut Creek, CA); Murphy, Michael B. (Severna Park, MD)

    2012-01-31

    The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

  10. Architecture of high-affinity unnatural-base DNA aptamers toward pharmaceutical applications

    OpenAIRE

    Ken-ichiro Matsunaga; Michiko Kimoto; Charlotte Hanson; Michael Sanford; Young, Howard A.; Ichiro Hirao

    2015-01-01

    We present a remodeling method for high-affinity unnatural-base DNA aptamers to augment their thermal stability and nuclease resistance, for use as drug candidates targeting specific proteins. Introducing a unique mini-hairpin DNA provides robust stability to unnatural-base DNA aptamers generated by SELEX using genetic alphabet expansion, without reducing their high affinity. By this method, >80% of the remodeled DNA aptamer targeting interferon-γ (K D of 33 pM) survived in human serum at 37 ...

  11. Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer

    DEFF Research Database (Denmark)

    Pasternak, Anna; Hernandez, Frank J; Rasmussen, Lars Melholt;

    2011-01-01

    A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA...... that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties...

  12. Nucleic Acid Aptamers as Novel Class of Therapeutics to Mitigate Alzheimer's Disease Pathology

    DEFF Research Database (Denmark)

    K. Tannenberg, Rudi; Al. Shamaileh, Hadi; Lauridsen, Lasse Holm;

    2013-01-01

    with a goal of early detection of amyloid aggregation and the neutralization of A toxicity. Short single-stranded oligonucleotide aptamers bind with high affinity and specificity to their targets. Aptamers that specifically bind to A beta monomers, specifically the 40 and 42 amino acid species (A beta......(1-40) and A beta(1-42)), fibrils and plaques have a great potential for diagnostic applications and the treatment of AD. Herein, we review the aptamers that bind to the various forms of A beta peptides for use in diagnosis and to inhibit plaque formation....

  13. 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...... Nafion-coated membrane. The aptasensor response time was <1 s, and the sensitivity of analysis was 62 nA μM–1 cm–2. The proposed design of the aptasensor, based on electrostatic interactions between the positively charged cysteamine-modified electrode and the negatively charged aptamer, may be used as a...

  14. Aptamers and methods for their in vitro selection and uses thereof

    Science.gov (United States)

    Doyle, Sharon A.; Murphy, Michael B.

    2008-02-12

    The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

  15. Toxic effects of pesticide mixtures at a molecular level: Their relevance to human health

    International Nuclear Information System (INIS)

    Highlights: ► Toxic effects of pesticide mixtures can be independent, dose addition or interaction. ► Metabolic interactions involve inhibition or induction of detoxifying enzymes. ► Organophosphates can potentiate pyrethroid, carbaryl and triazine toxicity. ► Synergism occurs when two active pesticides elicit greater than additive toxicity. ► Endocrine disruptors have the potential for additivity rather than synergism. - Abstract: Pesticides almost always occur in mixtures with other ones. The toxicological effects of low-dose pesticide mixtures on the human health are largely unknown, although there are growing concerns about their safety. The combined toxicological effects of two or more components of a pesticide mixture can take one of three forms: independent, dose addition or interaction. Not all mixtures of pesticides with similar chemical structures produce additive effects; thus, if they act on multiple sites their mixtures may produce different toxic effects. The additive approach also fails when evaluating mixtures that involve a secondary chemical that changes the toxicokinetics of the pesticide as a result of its increased activation or decreased detoxification, which is followed by an enhanced or reduced toxicity, respectively. This review addresses a number of toxicological interactions of pesticide mixtures at a molecular level. Examples of such interactions include the postulated mechanisms for the potentiation of pyrethroid, carbaryl and triazine herbicides toxicity by organophosphates; how the toxicity of some organophosphates can be potentiated by other organophosphates or by previous exposure to organochlorines; the synergism between pyrethroid and carbamate compounds and the antagonism between triazine herbicides and prochloraz. Particular interactions are also addressed, such as those of pesticides acting as endocrine disruptors, the cumulative toxicity of organophosphates and organochlorines resulting in estrogenic effects and the

  16. Molecular Tools for the Selective Detection of Nine Diatom Species Biomarkers of Various Water Quality Levels

    Directory of Open Access Journals (Sweden)

    Lucia Cimarelli

    2015-05-01

    Full Text Available Our understanding of the composition of diatom communities and their response to environmental changes is currently limited by laborious taxonomic identification procedures. Advances in molecular technologies are expected to contribute more efficient, robust and sensitive tools for the detection of these ecologically relevant microorganisms. There is a need to explore and test phylogenetic markers as an alternative to the use of rRNA genes, whose limited sequence divergence does not allow the accurate discrimination of diatoms at the species level. In this work, nine diatom species belonging to eight genera, isolated from epylithic environmental samples collected in central Italy, were chosen to implement a panel of diatoms covering the full range of ecological status of freshwaters. The procedure described in this work relies on the PCR amplification of specific regions in two conserved diatom genes, elongation factor 1-a (eEF1-a and silicic acid transporter (SIT, as a first step to narrow down the complexity of the targets, followed by microarray hybridization experiments. Oligonucleotide probes with the potential to discriminate closely related species were designed taking into account the genetic polymorphisms found in target genes. These probes were tested, refined and validated on a small-scale prototype DNA chip. Overall, we obtained 17 highly specific probes targeting eEF1-a and SIT, along with 19 probes having lower discriminatory power recognizing at the same time two or three species. This basic array was validated in a laboratory setting and is ready for tests with crude environmental samples eventually to be scaled-up to include a larger panel of diatoms. Its possible use for the simultaneous detection of diatoms selected from the classes of water quality identified by the European Water Framework Directive is discussed.

  17. A framework for embedding molecular-level information in continuum-scale simulations of interfacial flows

    Science.gov (United States)

    Smith, Edward; Theodorakis, Panagiotis; Muller, Erich; Craster, Richard; Matar, Omar

    2015-11-01

    Molecular dynamics provides a means of resolving the contact-line paradox. The price to pay for this insight is computational, with droplet simulations limited to the nanoscale. In order to model problems of engineering interest, the molecular contact line must be abstracted and included as part of a continuum scale simulation. Coupling, using dynamic molecular simulation in place of empirical or approximate closure relations, provides a means of doing just this. Molecular simulation of two phase Couette flow can reproduce the key features of the moving contact line. This sheared liquid bridge has the advantage that a steady state can be obtained, providing an unlimited source of data for statistical analysis. In this talk, we will present highlights from molecular dynamics simulation of the moving contact line. Using interface tracking, the dynamics of the contact line are examined, with results compared to published experimental studies. Good agreement is observed despite the difference in scale between the molecular model and experiments. Potential applications of this method are discussed, including coupled simulation which incorporates the molecular detail for surfactant-driven spreading. EPSRC Platform Grant (MACIPh) EP/L020564/1.

  18. Molecular level mechanisms of quartz dissolution at neutral and alkaline conditions with the presence of electrolytes

    Science.gov (United States)

    Liu, Y.; Zhang, S.

    2012-12-01

    The mechanisms of quartz dissolution are intricately affected by pH and electrolyte types. While most of previous studies have focused on mechanisms of quartz dissolution under a single specific condition (e.g., temperature, pH, saturation, or electrolyte type), this study investigates the molecular level mechanisms at combinations of electrolyte and pH conditions, which are more complicated but closer to the reality. Under neutral and alkaline pH conditions, with one of the Ca2+, Mg2+ or Na+ electrolytes in the solution, the dissolution of Q1(Si) and Q2(Si) sites on quartz surface, which represents the most important part of the quartz dissolution story, were investigated by first-principles quantum chemistry calculation methods. Also, large cluster models were used to represent the surface structures of quartz. The M05-2X/6-311+G** level DFT (Density Functional Theory) calculations and the STQN (Synchronous Transit-Guided Quasi-Newton) method (i.e., the QST3 method in Gaussian 03) were used to search transition-state structures and calculate energy barriers of the elementary Si-O bond breaking steps. Our results confirm that the dissolution of quartz can be significantly enhanced with the presence of electrolytes under neutral pH conditions, while under alkaline pH conditions, the behaviors of electrolytes are complicated, depending on where and how the electrolytes bond to quartz surfaces. Under neutral conditions, almost all types of electrolytes can directly bond to the bridging oxygen (BO) sites, leading to a weakened Si-Obr bonding and an increase of quartz dissolution. At alkaline conditions, however, electrolytes can no longer link to BO sites but rather link to terminal oxygen sites, leading to different dissolution mechanisms of quartz. The behaviors of specific electrolytes Na+, Ca2+, and Mg2+ on Q1(Si) and Q2 (Si) sites are also different, leading to more complicated dissolution mechanisms. Finally, the calculated energy barriers of possible hydrolysis

  19. Screening and Initial Binding Assessment of Fumonisin B1 Aptamers

    Directory of Open Access Journals (Sweden)

    Maria C. DeRosa

    2010-11-01

    Full Text Available Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize. Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC. The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B1. Six unique sequences were obtained, each showing improved binding to fumonisin B1 compared to controls. Sequence FB1 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.

  20. Determination of endotoxin through an aptamer-based impedance biosensor.

    Science.gov (United States)

    Su, Wenqiong; Lin, Meng; Lee, Hyuck; Cho, MiSuk; Choe, Woo-Seok; Lee, Youngkwan

    2012-02-15

    Lipopolysaccharide (LPS) often referred to endotoxin is an undesirable impurity frequently entrained with various recombinant protein therapeutics and plasmid DNA (pDNA) vaccines of bacterial origin. The inherent toxicities (e.g. fever, hypotension, shock and death) of LPS render its early and sensitive detection essential for several biological assays and/or parenteral administrations of biotherapeutics. In this study, an electrochemical biosensor using an LPS specific single stranded DNA (ssDNA) aptamer as a probe was developed. Amine-terminated aptamer exhibiting high affinity (K(d)=11.9 nM) to LPS was immobilized on a gold electrode using 3-mercaptopropionic acid (MPA) as a linker. Each step of the modification process was characterized by cyclic voltammetry (CV) and electrochemical impendence spectroscopy (EIS). A good linear relationship of the changes in the charge-transfer resistance (ΔR(et)) and the logarithmic value of LPS concentration was demonstrated in a broad dynamic detection range of 0.001-1 ng/ml. Furthermore, the aptasensor showed a high selectivity to LPS despite the presence of pDNA, RNA and bovine serum albumin (BSA) and could be regenerated in low pH condition, offering a promising option for detecting LPS often present in a complex milieu. PMID:22182428

  1. Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity

    OpenAIRE

    Chiu, Isaac M; Barrett, Lee B.; Williams, Erika K.; Strochlic, David E; Lee, Seungkyu; Weyer, Andy D.; Lou, Shan; Bryman, Gregory S; Roberson, David P.; Ghasemlou, Nader; Piccoli, Cara; Ahat, Ezgi; Wang, Victor; Cobos del Moral, Enrique Jos??; Cheryl L. Stucky

    2014-01-01

    The somatosensory nervous system is critical for the organism's ability to respond to mechanical, thermal, and nociceptive stimuli. Somatosensory neurons are functionally and anatomically diverse but their molecular profiles are not well-defined. Here, we used transcriptional profiling to analyze the detailed molecular signatures of dorsal root ganglion (DRG) sensory neurons. We used two mouse reporter lines and surface IB4 labeling to purify three major non-overlapping classes of neurons: 1)...

  2. Molecular-Level Transformations of Lignin During Photo-Oxidation and Biodegradation

    Science.gov (United States)

    Feng, X.; Hills, K.; Simpson, A. J.; Simpson, M. J.

    2009-05-01

    As the second most abundant component of terrestrial plant residues, lignin plays a key role in regulating plant litter decomposition, humic substance formation, and dissolved organic matter (OM) production from terrestrial sources. Biodegradation is the primary decomposition process of lignin on land. However, photo- oxidation of lignin-derived compounds has been reported in aquatic systems and is considered to play a vital role in arid and semiarid regions. With increasing ultraviolet (UV) radiation due to ozone depletion, it is important to understand the biogeochemical fate of lignin exposed to photo-oxidation in terrestrial environments. This study examines and compares the transformation of lignin in a three-month laboratory simulation of biodegradation and photo-oxidation using molecular-level techniques. Lignin-derived monomers extracted by copper oxidation were analyzed by gas chromatography/mass spectrometry (GC/MS) from the water-soluble and insoluble OM of 13C-labeled corn leaves. Biodegradation increased the solubility of lignin monomers in comparison to the control samples, and the acid-to-aldehyde (Ad/Al) ratios increased in both the water-soluble and insoluble OM, indicating a higher degree of side-chain lignin oxidation. Photo-oxidation did not produce a significant change on the solubility or Ad/Al ratios of lignin from corn leaves. However, the ratios of trans-to-cis isomers of both cinnamyl units (p-coumaric acid and ferulic acid) increased with photo-oxidation and decreased with biodegradation in the insoluble OM. We also investigated the role of photo-oxidation in lignin transformation in soils cropped with 13C-labeled corn. Interestingly, the organic carbon content increased significantly with time in the water-soluble OM from soil/corn residues under UV radiation. An increase in the concentration of lignin monomers and dimers and the Ad/Al ratios was also observed with photo-oxidation. Iso-branched fatty acids of microbial origin remained in

  3. Fluorescence correlation spectroscopy of gold nanoparticles, and its application to an aptamer-based homogeneous thrombin assay

    International Nuclear Information System (INIS)

    We have studied the fluorescence properties and diffusion behaviors of gold nanoparticles (GNPs) in solution by using fluorescence correlation spectroscopy (FCS) at single molecule level. The GNPs display a high photo-saturation feature. Under illumination with strong laser light, they display higher brightness per particle (BPP) despite their low quantum yields. Based on the unique fluorescence properties and diffusion behaviors of GNPs, we have developed a sensitive and homogenous thrombin assay. It is based on a sandwich strategy and is making use of GNPs to which two different aptamers are conjugated. When the differently aptamer-labeled GNPs are mixed with solutions containing thrombin, the affinity reaction causes the GNPs to form dimers or oligomers. This leads to an increase in the diffusion time of the GNPs in the detection volume that is seen in FCS. The FCS method enables sensitive detection of the change in the characteristic diffusion time of the GNPs before and after the affinity reaction. Quantitative analysis of thrombin is based on the measurement of the change in the diffusion time. Under optimal conditions, the calibration plot is linear in the 0.5 nM to 110 nM thrombin concentration range, and the detection limit is 0.5 nM. The method was successfully applied to the direct determination of thrombin in human plasma. (author)

  4. Aptamers and Capillary Electrophoresis: physico-chemical characterization of free aptamers in solution or grafted on nanoparticles, and study of their affinity towards a proteic target in view of their use in sensitive diagnosis methods

    OpenAIRE

    Girardot, Marie

    2010-01-01

    Aptamers are short oligonucleotides selected by SELEX (systematic evolution of ligands by exponential enrichment) and showing great affinity and specificity towards their target. This work deals with the physico-chemical characterization of aptamers and the study of their affinity towards their target, using capillary electrophoresis, through the example of an aptamer selected against a highly basic protein, lysozyme. After having evaluated several modifications of the capillary surface, the ...

  5. Fluorescent assay for oxytetracycline based on a long-chain aptamer assembled onto reduced graphene oxide

    International Nuclear Information System (INIS)

    We report on a fluorescent assay for oxytetracycline (OTC) using a fluorescein-labeled long-chain aptamer assembled onto reduced graphene oxide (rGO). The π-π stacking interaction between aptamer and rGO causes the fluorescence of the label to be almost completely quenched via energy transfer so that the system has very low background fluorescence. The addition of OTC leads to the formation of G-quadruplex OTC complexes and prevents the adsorption of labeled aptamer on the surface of rGO. As a result, fluorescence is restored, and this effect allows for a quantitative assay of OTC over the 0.1–2 μM concentration range and with a detection limit of 10 nM. This method is simple, rapid, selective and sensitive. It may be applied to other small molecule analytes by applying appropriate aptamers. (author)

  6. 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

    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...... 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...

  7. Aptamer-based radiopharmaceuticals for diagnostic imaging and targeted radiotherapy of epithelial tumors

    Energy Technology Data Exchange (ETDEWEB)

    Missailidis, Sotiris [The Open University, Milton Keynes (United Kingdom). Dept. of Chemistry and Analytical Sciences]. E-mail: s.missailidis@open.ac.uk; Perkins, Alan [University of Nottingham (United Kingdom). Dept. of Medical Physics; Santos-Filho, Sebastiao David; Fonseca, Adenilson de Souza da; Bernardo-Filho, Mario [Universidade do Estado do Rio de Janeiro (UERJ), RJ (Brazil). Inst. de Biologia Roberto Alcantara Gomes. Dept. de Biofisica e Biometria

    2008-12-15

    In the continuous search for earlier diagnosis and improved therapeutic modalities against cancer, based on our constantly increasing knowledge of cancer biology, aptamers hold the promise to expand on current antibody success, but overcoming some of the problems faced with antibodies as therapeutic or delivery agents in cancer. However, as the first aptamer reached the market as an inhibitor against angiogenesis for the treatment of macular degeneration, aptamers have found only limited applications or interest in oncology, and even less as radiopharmaceuticals for diagnostic imaging and targeted radiotherapy of tumours. Yet, the chemistry for the labelling of aptamers and the options to alter their pharmacokinetic properties, to make them suitable for use as radiopharmaceuticals is now available and recent advances in their development can demonstrate that these molecules would make them ideal delivery vehicles for the development of targeted radiopharmaceuticals that could deliver their radiation load with accuracy to the tumour site, offering improved therapeutic properties and reduced side effects. (author)

  8. Selection of DMA aptamer that specific binding human carcinoembryonic antigen in vitro

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective:To select the specific aptamer of carcinoembryonic antigen (CEA), one of the most attractive molecule for cancer target therapy and imaging. Methods: Seven rounds in vitro selection were performed against the purified CEA protein. Ligand-mediated target purification and Co-immunoprecipitation were adopted to verify the specific binding of the aptamer to the purified and native protein separately. Results:The CEA-specific aptamer which can bind both the purified and native protein with the high specificity was obtained. Conclusion:This is the first time the CEA specific apatmer was produced. The results in this study provides the preliminary evidence for further investigation and application of CEA-aptamer in the future.

  9. Development of an aptamer-conjugated fluorescent nanoprobe for MMP2

    Science.gov (United States)

    Han, Myoung-Eun; Baek, Sungmin; Kim, Hyun-Jung; Lee, Jung Hwan; Ryu, Sung-Ho; Oh, Sae-Ock

    2014-03-01

    Matrix metalloproteinase 2 (MMP2) plays critical roles in various diseases, such as atherosclerosis and cancer, and has been suggested to contribute to the instability of atherosclerotic plaque. To visualize MMP2 in pathologic tissues, we developed an aptamer targeting MMP2 protein by performing eight rounds of modified DNA systematic evolution of ligands by exponential enrichment (SELEX). The aptamer showed high affinity for MMP2 ( K d = 5.59 nM), precipitated MMP2, and detected MMP2 protein in pathological tissues such as atherosclerotic plaque and gastric cancer tissues. Furthermore, a MMP2 aptamer-conjugated fluorescent nanoprobe successfully visualized atherosclerotic plaques in apolipoprotein E (ApoE) knockout mice. These results suggest that the devised MMP2 aptamer could be useful for the development of various diagnostic tools.

  10. Aptamer-based radiopharmaceuticals for diagnostic imaging and targeted radiotherapy of epithelial tumors

    International Nuclear Information System (INIS)

    In the continuous search for earlier diagnosis and improved therapeutic modalities against cancer, based on our constantly increasing knowledge of cancer biology, aptamers hold the promise to expand on current antibody success, but overcoming some of the problems faced with antibodies as therapeutic or delivery agents in cancer. However, as the first aptamer reached the market as an inhibitor against angiogenesis for the treatment of macular degeneration, aptamers have found only limited applications or interest in oncology, and even less as radiopharmaceuticals for diagnostic imaging and targeted radiotherapy of tumours. Yet, the chemistry for the labelling of aptamers and the options to alter their pharmacokinetic properties, to make them suitable for use as radiopharmaceuticals is now available and recent advances in their development can demonstrate that these molecules would make them ideal delivery vehicles for the development of targeted radiopharmaceuticals that could deliver their radiation load with accuracy to the tumour site, offering improved therapeutic properties and reduced side effects. (author)

  11. Molecular Processes Studied at a Single-Molecule Level Using DNA Origami Nanostructures and Atomic Force Microscopy

    Directory of Open Access Journals (Sweden)

    Ilko Bald

    2014-09-01

    Full Text Available DNA origami nanostructures allow for the arrangement of different functionalities such as proteins, specific DNA structures, nanoparticles, and various chemical modifications with unprecedented precision. The arranged functional entities can be visualized by atomic force microscopy (AFM which enables the study of molecular processes at a single-molecular level. Examples comprise the investigation of chemical reactions, electron-induced bond breaking, enzymatic binding and cleavage events, and conformational transitions in DNA. In this paper, we provide an overview of the advances achieved in the field of single-molecule investigations by applying atomic force microscopy to functionalized DNA origami substrates.

  12. Toggled RNA Aptamers Against Aminoglycosides Allowing Facile Detection of Antibiotics Using Gold Nanoparticle Assays

    OpenAIRE

    Derbyshire, Nicola; White, Simon J.; Bunka, David H. J.; Song, Lei; Stead, Sara; Tarbin, Jonathan; Sharman, Matthew; Zhou, Dejian; Stockley, Peter G.

    2012-01-01

    We have used systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers against aminoglycoside antibiotics. The SELEX rounds were toggled against four pairs of aminoglycosides with the goal of isolating reagents that recognize conserved structural features. The resulting aptamers bind both of their selection targets with nanomolar affinities. They also bind the less structurally related targets, although they show clear specificity for this class of antibiotics....

  13. Nucleic Acid Aptamers: An Emerging Tool for Biotechnology and Biomedical Sensing

    Directory of Open Access Journals (Sweden)

    Ti-Hsuan Ku

    2015-07-01

    Full Text Available Detection of small molecules or proteins of living cells provides an exceptional opportunity to study genetic variations and functions, cellular behaviors, and various diseases including cancer and microbial infections. Our aim in this review is to give an overview of selected research activities related to nucleic acid-based aptamer techniques that have been reported in the past two decades. Limitations of aptamers and possible approaches to overcome these limitations are also discussed.

  14. Efficient reverse transcription using locked nucleic acid nucleotides towards the evolution of nuclease resistant RNA aptamers

    DEFF Research Database (Denmark)

    Crouzier, Lucile; Dubois, Camille; Edwards, Stacey L;

    2012-01-01

    We found that SuperScript® III Reverse Transcriptase is an efficient enzyme for the recognition of LNA nucleotides, making it a prime candidate to be used in de novo selection of LNA containing RNA aptamers.......We found that SuperScript® III Reverse Transcriptase is an efficient enzyme for the recognition of LNA nucleotides, making it a prime candidate to be used in de novo selection of LNA containing RNA aptamers....

  15. Aptamers as targeting delivery devices or anti-cancer drugs for fighting tumors.

    Science.gov (United States)

    Scaggiante, Bruna; Dapas, Barbara; Farra, Rossella; Grassi, Mario; Pozzato, Gabriele; Giansante, Carlo; Fiotti, Nicola; Tamai, Elisa; Tonon, Federica; Grassi, Gabriele

    2013-06-01

    Aptamer researches applied to the treatment of human cancers have increased since their discovery in 1990. This is due to different factors including: 1) the technical possibility to select, by SELEX-based procedures, specific aptamers targeting virtually any given molecule, 2) the aptamer favorable bio-activity in vivo, 3) the low production costs and 4) the ease synthesis and storage for the marketing. In the field of cancer treatments, aptamers have been studied as tumor-specific agents driving drugs into cancer cells; additionally they have been used as anti-neoplastic agents, able to inhibit tumor cell growth and dissemination when administered alone or in combination with conventional anti-neoplastic drugs. Aptamers are gaining an increased interest for pharmaceutical companies and some of them are under clinical evaluation trials. In this review we update the findings about the use of aptamers as "escort" molecules able to drive drugs into the cells and as antineoplastic drugs. Current anti-neoplastic treatments suffer from the intrinsic toxicity related to the un-specific targeting of both normal and tumorigenic proliferating cells. The aptamers could be useful to improve: 1) the selective targeting of molecules essential for the viability and expansion of tumor cells and/or the selective driving of chemotherapies into tumor cells, thus resulting in higher effectiveness and lower systemic side-effects compared to conventional anti-neoplastic drugs alone and 2) to improve the therapeutic index of currently used chemotherapies. Even if some problems related to the in vivo stability and pharmacokinetic/dynamics of aptamers remain to be improved, their potential use in the treatment of different human cancers is getting closer and closer to a practical therapeutic use. PMID:23687927

  16. Direct visualization of electrophoretic mobility shift assays using nanoparticle-aptamer conjugates

    OpenAIRE

    Wang, Min S.; Reed, Scott M.

    2011-01-01

    Here we demonstrate that aptamers tethered to gold nanoparticles enable direct visualization of protein-oligonucleotide interactions during gel electrophoresis. This technique is used to confirm that an aptamer previously identified as binding to C-reactive protein (CRP) only binds to the monomeric form of CRP. While native, pentameric CRP (pCRP) is used in clinical assays to predict cardiovascular disease (CVD) risk, it is the monomeric isoform that is more strongly associated with pro-infla...

  17. Aptamer conjugated paclitaxel and magnetic fluid loaded fluorescently tagged PLGA nanoparticles for targeted cancer therapy

    Science.gov (United States)

    Aravind, Athulya; Nair, Remya; Raveendran, Sreejith; Veeranarayanan, Srivani; Nagaoka, Yutaka; Fukuda, Takahiro; Hasumura, Takahashi; Morimoto, Hisao; Yoshida, Yasuhiko; Maekawa, Toru; Sakthi Kumar, D.

    2013-10-01

    Controlled and targeted drug delivery is an essential criterion in cancer therapy to reduce the side effects caused by non-specific drug release and toxicity. Targeted chemotherapy, sustained drug release and optical imaging have been achieved using a multifunctional nanocarrier constructed from poly (D, L-lactide-co-glycolide) nanoparticles (PLGA NPs), an anticancer drug paclitaxel (PTX), a fluorescent dye Nile red (NR), magnetic fluid (MF) and aptamers (Apt, AS1411, anti-nucleolin aptamer). The magnetic fluid and paclitaxel loaded fluorescently labeled PLGA NPs (MF-PTX-NR-PLGA NPs) were synthesized by a single-emulsion technique/solvent evaporation method using a chemical cross linker bis (sulfosuccinimidyl) suberate (BS3) to enable binding of aptamer on to the surface of the nanoparticles. Targeting aptamers were then introduced to the particles through the reaction with the cross linker to target the nucleolin receptors over expressed on the cancer cell surface. Specific binding and uptake of the aptamer conjugated magnetic fluid loaded fluorescently tagged PLGA NPs (Apt-MF-NR-PLGA NPs) to the target cancer cells induced by aptamers was observed using confocal microscopy. Cytotoxicity assay conducted in two cell lines (L929 and MCF-7) confirmed that targeted MCF-7 cancer cells were killed while control cells were unharmed. In addition, aptamer mediated delivery resulting in enhanced binding and uptake to the target cancer cells exhibited increased therapeutic effect of the drug. Moreover, these aptamer conjugated magnetic polymer vehicles apart from actively transporting drugs into specifically targeted tumor regions can also be used to induce hyperthermia or for facilitating magnetic guiding of particles to the tumor regions.

  18. Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers.

    Science.gov (United States)

    Jolly, Pawan; Estrela, Pedro; Ladomery, Michael

    2016-06-30

    There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications. PMID:27365033

  19. Discovering Aptamers by Cell-SELEX against Human Soluble Growth Factors Ectopically Expressed on Yeast Cell Surface

    OpenAIRE

    Hsien-Wei Meng; Pagano, John M.; White, Brian S.; Yoshiko Toyoda; Min, Irene M.; Craighead, Harold G; David Shalloway; Lis, John T.; Kai Xiao; Moonsoo M Jin

    2014-01-01

    SELEX, the process of selecting aptamers, is often hampered by the difficulty of preparing target molecules in their native forms and by a lack of a simple yet quantitative assay for monitoring enrichment and affinity of reactive aptamers. In this study, we sought to discover DNA aptamers against human serum markers for potential therapeutic and diagnostic applications. To circumvent soluble expression and immobilization for performing SELEX, we ectopically expressed soluble growth factors on...

  20. Codeine-binding RNA aptamers and rapid determination of their binding constants using a direct coupling surface plasmon resonance assay

    OpenAIRE

    Win, Maung Nyan; Klein, Joshua S.; Smolke, Christina D.

    2006-01-01

    RNA aptamers that bind the opium alkaloid codeine were generated using an iterative in vitro selection process. The binding properties of these aptamers, including equilibrium and kinetic rate constants, were determined through a rapid, high-throughput approach using surface plasmon resonance (SPR) analysis to measure real-time binding. The approach involves direct coupling of the target small molecule onto a sensor chip without utilization of a carrier protein. Two highest binding aptamer se...

  1. Surface-Enhanced Raman Spectroscopy Based Quantitative Bioassay on Aptamer-Functionalized Nanopillars Using Large-Area Raman Mapping

    DEFF Research Database (Denmark)

    Yang, Jaeyoung; Palla, Mirko; Bosco, Filippo;

    2013-01-01

    functionalized with aptamers for sensitive and specific detection of target molecules. In this study, TAMRA-labeled vasopressin molecules in the picomolar regime (1 pM to 1 nM) are specifically captured by aptamers on the nanostructured SERS substrate and monitored by using an automated SERS signal mapping...... reliable analysis of each measurement. Combining our SERS mapping analysis with an aptamer-functionalized nanopillar substrate is found to be extremely efficient for detection of low-abundance biomolecules....

  2. Probing high-affinity 11-mer DNA aptamer against Lup an 1 (β-conglutin).

    Science.gov (United States)

    Nadal, P; Svobodova, M; Mairal, T; O'Sullivan, C K

    2013-11-01

    Aptamers are synthetic nucleic acids with great potential as analytical tools. However, the length of selected aptamers (typically 60-100 bases) can affect affinity, due to the presence of bases not required for interaction with the target, and therefore, the truncation of these selected sequences and identification of binding domains is a critical step to produce potent aptamers with higher affinities and specificities and lowered production costs. In this paper we report the truncation of an aptamer that specifically binds to β-conglutin (Lup an 1), an anaphylactic allergen. Through comparing the predicted secondary structures of the aptamers, a hairpin structure with a G-rich loop was determined to be the binding motif. The highest affinity was observed with a truncation resulting in an 11-mer sequence that had an apparent equilibrium dissociation constant (K D) of 1.7 × 10(-9) M. This 11-mer sequence was demonstrated to have high specificity for β-conglutin and showed no cross-reactivity to other lupin conglutins (α-, δ-, γ-conglutins) and closely related proteins such as gliadin. Finally, the structure of the truncated 11-mer aptamer was preliminarily elucidated, and the GQRS Mapper strongly predicted the presence of a G-quadruplex, which was subsequently corroborated using one-dimensional NMR, thus highlighting the stability of the truncated structure. PMID:24126837

  3. 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.

  4. Detection of Cryptosporidium parvum Oocysts on Fresh Produce Using DNA Aptamers.

    Directory of Open Access Journals (Sweden)

    Asma Iqbal

    Full Text Available There are currently no standard methods for the detection of Cryptosporidium spp., or other protozoan parasites, in foods, and existing methods are often inadequate, with low and variable recovery efficiencies. Food testing is difficult due to the low concentrations of parasites, the difficulty in eluting parasites from some foods, the lack of enrichment methods, and the presence of PCR inhibitors. The main objectives of the present study were to obtain DNA aptamers binding to the oocyst wall of C. parvum, and to use the aptamers to detect the presence of this parasite in foods. DNA aptamers were selected against C. parvum oocysts using SELEX (Systematic Evolution of Ligands by EXponential enrichment. Ten rounds of selection led to the discovery of 14 aptamer clones with high affinities for C. parvum oocysts. For detecting parasite-bound aptamers, a simple electrochemical sensor was employed, which used a gold nanoparticle-modified screen-printed carbon electrode. This aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and the anti- C. parvum aptamer. Square wave voltammetry was employed to quantitate C. parvum in the range of 150 to 800 oocysts, with a detection limit of approximately 100 oocysts. The high sensitivity and specificity of the developed aptasensor suggests that this novel method is very promising for the detection and identification of C. parvum oocysts on spiked fresh fruits, as compared to conventional methods such as microscopy and PCR.

  5. The fabrication, characterization and application of aptamer-functionalized Si-nanowire FET biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ki Su; Lee, Hyun-Seung; Yang, Jeong-A; Jo, Moon-Ho; Hahn, Sei Kwang [Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), San 31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784 (Korea, Republic of)], E-mail: skhanb@postech.ac.kr

    2009-06-10

    An aptamer-functionalized silicon-nanowire (Si-NW) field effect transistor (FET) biosensor was successfully fabricated, characterized and applied to real-time electrical detection of binding with the target protein for biomedical applications. Surface modifications were carried out using 3-aminopropyl diethoxysilane and succinic anhydride to introduce amine and carboxyl groups onto Si substrates. Anti-thrombin aptamers with 5{sup '}-end amine groups were chemically grafted onto the surface-modified Si substrates through amide bond formation. Atomic force microscopic (AFM) analyses confirmed the successful immobilization of anti-thrombin aptamers on Si-NWs and their binding with thrombin samples. The anti-thrombin aptamers bound to Si-NWs through the linker appeared to have a mean height of approx. 4 nm and the thrombin/aptamer complex to have a mean height of approx. 8 nm. Fluorescence micrographs visualized the FITC-labeled thrombin after binding to anti-thrombin aptamers immobilized on Si-NWs. Furthermore, the anti-thrombin Si-NW FET biosensor was successfully applied to the real-time detection of electronic signals during and after binding with a thrombin sample at a concentration of approx. 330 pmol l{sup -1} and the thrombin in blood samples.

  6. Aptamer from whole-bacterium SELEX as new therapeutic reagent against virulent Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death. One-third of the world's population is infected with Mycobacterium tuberculosis (MTB), the etiologic agent of TB. Because of the global health problems of TB, the development of potent new anti-TB drugs without cross-resistance with known antimycobacterial agents is urgently needed. In this study, we have applied a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process to identify a single aptamer (NK2) that binds to virulent strain M. tuberculosis (H37Rv) with high affinity and specificity. We have found that this aptamer improves CD4+T cells to produce IFN-γ after binding to H37Rv. The different component between H37Rv and BCG was identified as some membrane protein. Moreover, the survival rates of mice challenged with i.v. H37Rv have been prolonged after treatment with single injection of aptamer NK2. The bacterial numbers were significantly lower in the spleen of mice treated with aptamer NK2. The histopathological examination of lung biopsy specimens showed lesser pulmonary alveolar fusion and swelling in the presence of the aptamer. These results suggest that aptamer NK2 has inhibitory effects on M. tuberculosis and can be used as antimycobacterial agent

  7. 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.

  8. Functional genomics in fish towards understanding stress and immune responses at a molecular level /

    OpenAIRE

    Ribas Cabezas, Laia

    2006-01-01

    Aquesta tesis doctoral està basada en estudiar la resposta immunològica dels peixos en models d'estrès i d'activació del sistema immune des la genòmica funcional. L'aplicació de tecnologies moleculars com el Differential Display van permetre identificar y clonar por primera vegada en orades (Sparus aurata) y en altres especies de peix, el gen enolasa. Aquest enzim glucolític s'ha plantejat per primera vegada com un bon marcador molecular per estudiar el benestar dels peixos. Per mitjà de l'ús...

  9. N-Heterocyclic Carbene-Gold(I) Complexes Conjugated to a Leukemia-Specific DNA Aptamer for Targeted Drug Delivery.

    Science.gov (United States)

    Niu, Weijia; Chen, Xigao; Tan, Weihong; Veige, Adam S

    2016-07-25

    This report describes the synthesis and characterization of novel N-heterocyclic carbene (NHC)-gold(I) complexes and their bioconjugation to the CCRF-CEM-leukemia-specific aptamer sgc8c. Successful bioconjugation was confirmed by the use of fluorescent tags on both the NHC-Au(I) complex and the aptamer. Cell-viability assays indicated that the NHC-Au(I) -aptamer conjugate was more cytotoxic than the NHC-gold complex alone. A combination of flow cytometry, confocal microscopy, and cell-viability assays provided clear evidence that the NHC-Au(I) -aptamer conjugate was selective for targeted CCRF-CEM leukemia cells. PMID:27311814

  10. Thrombin-linked aptamer assay for detection of platelet derived growth factor BB on magnetic beads in a sandwich format.

    Science.gov (United States)

    Guo, Limin; Zhao, Qiang

    2016-09-01

    Here we describe a thrombin-linked aptamer assay (TLAA) for protein by using thrombin as an enzyme label, harnessing enzyme activity of thrombin and aptamer affinity binding. TLAA converts detection of specific target proteins to the detection of thrombin by using a DNA sequence that consists of two aptamers with the first aptamer binding to the specific target protein and the second aptamer binding to thrombin. Through the affinity binding, the thrombin enzyme is labeled on the protein target, and thrombin catalyzes the hydrolysis of small peptide substrate into product, generating signals for quantification. As a proof of principle, we show a sandwich TLAA for platelet derived growth factor BB (PDGF-BB) by using anti-PDGF-BB antibody coated on magnetic beads and an oligonucleotide containing the aptamer for PDGF-BB and the aptamer for thrombin. The binding of PDGF-BB to both the antibody and the aptamer results in labeling the complex with thrombin. We achieved detection of PDGF-BB at 16 pM. This TLAA contributes a new application of thrombin and its aptamer in bioanalysis, and shows potentials in assay developments. PMID:27343590

  11. Molecular phylogeny of the Astrophorida (Porifera, Demospongiae) reveals an unexpected high level of spicule homoplasy

    OpenAIRE

    Paco Cárdenas; Xavier, Joana R.; Julie Reveillaud; Christoffer Schander; Hans Tore Rapp

    2011-01-01

    BackgroundThe Astrophorida (Porifera, Demospongiaep) is geographically and bathymetrically widely distributed. Systema Porifera currently includes five families in this order: Ancorinidae, Calthropellidae, Geodiidae, Pachastrellidae and Thrombidae. To date, molecular phylogenetic studies including Astrophorida species are scarce and offer limited sampling. Phylogenetic relationships within this order are therefore for the most part unknown and hypotheses based on morphology largely untested. ...

  12. Molecular-Level Simulation of Reacting Systems in Bulk and Confinement

    Czech Academy of Sciences Publication Activity Database

    Lísal, Martin; Smith, W. R.; Brennan, J. K.

    Praha : Process Engineering Publisher, 2004, s. 502. ISBN 80-86059-40-5. [International Congress of Chemical and Process Engineering CHISA 2004 /16./. Praha (CZ), 22.08.2004-26.08.2004] Institutional research plan: CEZ:AV0Z4072921 Keywords : molecular simulation * bulk and confinement * reacting systems Subject RIV: CF - Physical ; Theoretical Chemistry

  13. Dietary factors associated with plasma high molecular weight and total adiponectin levels in apparently healthy women

    NARCIS (Netherlands)

    Yannakoulia, Mary; Yiannakouris, Nikos; Melistas, Labros; Fappa, Evaggelia; Vidra, Nikoletta; Kontogianni, Meropi D; Mantzoros, Christos S

    2008-01-01

    OBJECTIVE: Our aim was to investigate associations between dietary factors and high molecular weight (HMW) as well as total adiponectin in a sample of apparently healthy adult Mediterranean women. DESIGN AND METHODS: Two hundred and twenty women were enrolled in this study. Anthropometric and body c

  14. Charge Transport in Molecular Junctions: A Study of Level-Alignment, Thermoelectric Properties, and Environmental Effects

    Science.gov (United States)

    Kotiuga, Michele

    Here, we use and develop first-principles methods based on density functional theory (DFT) and beyond to understand and predict charge transport phenomena in the novel class of nanostructured devices: molecular junctions. Molecular junctions, individual molecules contacted to two metallic leads, which can be systematically altered by modifying the chemistry of each component, serve as test beds for the study of transport at the nanoscale. To date, various experimental methods have been designed to reliably assemble and measure transport properties of molecular junctions. Furthermore, theoretical methods built on DFT designed to yield quantitative agreement with these experiments for certain classes of molecular junctions have been developed. In order to gain insight into a broader range of molecular junctions and environmental effects associated with the surrounding solution, this dissertation will employ, explore and extend first-principles DFT calculations coupled with approximate self-energy corrections known to yield quantitative agreement with experiments for certain classes of molecular junctions. To start we examine molecular junctions in which the molecule is strongly hybridized with the leads: a challenging limit for the existing methodology. Using a physically motivated tight-binding model, we find that the experimental trends observed for such molecules can be explained by the presence of a so-called "gateway" state associated with the chemical bond that bridges the molecule and the lead. We discuss the ingredients of a self-energy corrected DFT based approach to quantitatively predict conductance in the presence of these hybridization effects. We also develop and apply an approach to account for the surrounding environment on the conductance, which has been predominantly ignored in past transport calculations due to computational complexity. Many experiments are performed in a solution of non-conducting molecules; far from benign, this solution is known

  15. A fully quantal molecular description for the spectra of bosons and fermions in the lowest Landau level

    OpenAIRE

    Yannouleas, Constantine; Landman, Uzi

    2009-01-01

    Through the introduction of a class of appropriate translationally invariant trial wave functions, we show that the strong correlations in the lowest Landau level (LLL) reflect in finite systems the emergence of intrinsic point-group symmetries associated with rotations and vibrations of molecules formed through particle localization. This quantal molecular description is universal, being valid for both bosons and fermions, for both the yrast and excited states of the LLL spectra, and for bot...

  16. Programmatic Use of Molecular Xenomonitoring at the Level of Evaluation Units to Assess Persistence of Lymphatic Filariasis in Sri Lanka

    OpenAIRE

    Rao, Ramakrishna U.; Samarasekera, Sandhya D.; Kumara C Nagodavithana; Punchihewa, Manjula W.; Dassanayaka, Tharanga D. M.; Gamini P K D; Ethan Ford; Udaya S B Ranasinghe; Henderson, Ralph H.; Gary J Weil

    2016-01-01

    Background Sri Lanka’s Anti Filariasis Campaign distributed 5 rounds of mass drug administration (MDA with DEC plus albendazole) to all endemic regions in the country from 2002–2006. Post-MDA surveillance results have generally been encouraging. However, recent studies have documented low level persistence of Wuchereria bancrofti in Galle district based on comprehensive surveys that include molecular xenomonitoring (MX, detection of filarial DNA in mosquitoes) results. The purposes of this st...

  17. 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

  18. Advance of Molecular Imaging Technology and Targeted Imaging Agent in Imaging and Therapy

    Directory of Open Access Journals (Sweden)

    Zhi-Yi Chen

    2014-01-01

    Full Text Available Molecular imaging is an emerging field that integrates advanced imaging technology with cellular and molecular biology. It can realize noninvasive and real time visualization, measurement of physiological or pathological process in the living organism at the cellular and molecular level, providing an effective method of information acquiring for diagnosis, therapy, and drug development and evaluating treatment of efficacy. Molecular imaging requires high resolution and high sensitive instruments and specific imaging agents that link the imaging signal with molecular event. Recently, the application of new emerging chemical technology and nanotechnology has stimulated the development of imaging agents. Nanoparticles modified with small molecule, peptide, antibody, and aptamer have been extensively applied for preclinical studies. Therapeutic drug or gene is incorporated into nanoparticles to construct multifunctional imaging agents which allow for theranostic applications. In this review, we will discuss the characteristics of molecular imaging, the novel imaging agent including targeted imaging agent and multifunctional imaging agent, as well as cite some examples of their application in molecular imaging and therapy.

  19. Molecular-Level Processes Governing the Interaction of Contaminants with Iron and Manganese Oxides - Final Report; FINAL

    International Nuclear Information System (INIS)

    Many of the inorganic and organic contaminants present in sediments at DOE sites can be altered or destroyed by reduction and oxidation (redox) reactions occurring at mineral surfaces. A fundamental understanding of such redox processes provided by molecular-level studies on structurally and compositionally well-defined mineral surfaces will lead to: (i) improved models of contaminant fate and transport in geochemical systems, and (ii) optimized manipulation of these processes for remediation purposes. To contribute to this understanding, we will study, both experimentally and theoretically, redox processes involving three important contaminants - chromate ion, carbon tetrachloride, and trichloroethene TCE, on the following iron and manganese oxides - hematite, magnetite, maghemite, and pyrolusite. These oxides and their hydroxylated analogs commonly occur as coatings on minerals or as interfaces in the subsurface environment. Single-crystal surfaces of these oxides will be synthesized in carefully controlled fashion by molecular beam epitaxy. These surfaces, as well as high surface are powdered samples of these oxides, will be used in spectroscopic and kinetic experiments in both aqueous and gas phases. Our goal is to identify products and to determine the kinetics and mechanisms of surface-catalyzed redox reaction of Cr(VI) and CR(III), and the reductive dechlorination of carbon tetrachloride and TCE. The combination of theory and experiment will provide the base information needed to scale from the molecular level to the microscopic grain level minerals

  20. Spectroscopic investigations on the interactions between isopropanol and trypsin at molecular level

    Science.gov (United States)

    Hu, Xinxin; Yu, Zehua; Liu, Rutao

    2013-05-01

    The toxicity of hydroxyl group of isopropanol to trypsin in aqueous solution was investigated by techniques including UV-visible absorption spectroscopy, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, enzyme activity assay and molecular docking technology. The results of UV-visible absorption spectroscopy and CD spectra indicate that isopropanol could change the secondary structure of trypsin by increasing the content of α-helix and decreasing the content of β-sheet. The tertiary structure of trypsin was also changed owing to the loss of environmental asymmetry of amino acid residues. Isopropanol bound into a hydrophobic cavity on the surface of trypsin by a hydrogen bond located between the hydrogen atom on the hydroxyl of isopropanol and the oxygen atoms on SER 214 and hydrophobic interaction, as the molecular docking results showed. In addition, isopropanol could affect the function of trypsin by increasing its catalytic activity.

  1. Thickness control of molecular beam epitaxy grown layers at the 0.01–0.1 monolayer level

    International Nuclear Information System (INIS)

    Electron tunnelling through semiconductor tunnel barriers is exponentially sensitive to the thickness of the barrier layer, and in the most common system, the AlAs tunnel barrier in GaAs, a one monolayer variation in thickness results in a 300% variation in the tunnelling current for a fixed bias voltage. We use this degree of sensitivity to demonstrate that the level of control at 0.06 monolayer can be achieved in the growth by molecular beam epitaxy, and the geometrical variation of layer thickness across a wafer at the 0.01 monolayer level can be detected. (paper)

  2. Einstein–Bohr recoiling double-slit gedanken experiment performed at the molecular level

    OpenAIRE

    Liu, X-J; Miao, Q.; Gel'mukhanov, F.; Patanen, M.; Travnikova, O; C. Nicolas; Ågren, H; Ueda, K.; Miron, C.

    2015-01-01

    Double-slit experiments illustrate proof for wave–particle complementarity. The essence of Einstein–Bohr's debate about wave–particle duality was whether the momentum transfer between a particle and a recoiling slit could mark the path, thus destroying the interference. We realized this recoiling double-slit gedanken experiment by resonant X-ray photoemission from molecular oxygen for geometries near equilibrium (coupled slits) and in a dissociative state far away from equilibrium (decoupled ...

  3. Anthraquinone-based demultiplexer and other multiple operations at the molecular level

    Indian Academy of Sciences (India)

    Navneet Kaur; Subodh Kumar

    2014-01-01

    Anthraquinone-based chemosensor L with pyridine units as additional functional groups has been found to show pH-dependent multiple coordinationmodes towards differentmetal ions (Co2+, Ni2+ and Cu2+). Based on these different absorption changes, this differential colorimetric chemosensor L has found promising applications as a multiple-mode molecular logic system, i.e., OR, three - input NOR, three - input INHIBIT, TRANSFER and 1:2 DEMUX.

  4. Gene expression patterns unveil a new level of molecular heterogeneity in colorectal cancer

    OpenAIRE

    Budinska, Eva; Popovici, Vlad; Tejpar, Sabine; d'Ario, Giovanni; Lapique, Nicolas; Sikora, Katarzyna Otylia; Di Narzo, Antonio Fabio; Yan, Pu; Hodgson, John Graeme; Weinrich, Scott; Bosman, Fred; Roth, Arnaud; Delorenzi, Mauro

    2013-01-01

    The recognition that colorectal cancer (CRC) is a heterogeneous disease in terms of clinical behaviour and response to therapy translates into an urgent need for robust molecular disease subclassifiers that can explain this heterogeneity beyond current parameters (MSI, KRAS, BRAF). Attempts to fill this gap are emerging. The Cancer Genome Atlas (TGCA) reported two main CRC groups, based on the incidence and spectrum of mutated genes, and another paper reported an EMT expression signature defi...

  5. Gene expression patterns unveil a new level of molecular heterogeneity in colorectal cancer.

    OpenAIRE

    Budinska E.; Popovici V.; Tejpar S; D' Ario G.; Lapique N.; Sikora K.O.; Di Narzo A.F.; Yan P.; Hodgson J.G.; Weinrich S.; Bosman F.; Roth A.; Delorenzi M.

    2013-01-01

    The recognition that colorectal cancer (CRC) is a heterogeneous disease in terms of clinical behaviour and response to therapy translates into an urgent need for robust molecular disease subclassifiers that can explain this heterogeneity beyond current parameters (MSI, KRAS, BRAF). Attempts to fill this gap are emerging. The Cancer Genome Atlas (TGCA) reported two main CRC groups, based on the incidence and spectrum of mutated genes, and another paper reported an EMT expression signature defi...

  6. Gene expression patterns unveil a new level of molecular heterogeneity in colorectal cancer.

    Science.gov (United States)

    Budinska, Eva; Popovici, Vlad; Tejpar, Sabine; D'Ario, Giovanni; Lapique, Nicolas; Sikora, Katarzyna Otylia; Di Narzo, Antonio Fabio; Yan, Pu; Hodgson, John Graeme; Weinrich, Scott; Bosman, Fred; Roth, Arnaud; Delorenzi, Mauro

    2013-09-01

    The recognition that colorectal cancer (CRC) is a heterogeneous disease in terms of clinical behaviour and response to therapy translates into an urgent need for robust molecular disease subclassifiers that can explain this heterogeneity beyond current parameters (MSI, KRAS, BRAF). Attempts to fill this gap are emerging. The Cancer Genome Atlas (TGCA) reported two main CRC groups, based on the incidence and spectrum of mutated genes, and another paper reported an EMT expression signature defined subgroup. We performed a prior free analysis of CRC heterogeneity on 1113 CRC gene expression profiles and confronted our findings to established molecular determinants and clinical, histopathological and survival data. Unsupervised clustering based on gene modules allowed us to distinguish at least five different gene expression CRC subtypes, which we call surface crypt-like, lower crypt-like, CIMP-H-like, mesenchymal and mixed. A gene set enrichment analysis combined with literature search of gene module members identified distinct biological motifs in different subtypes. The subtypes, which were not derived based on outcome, nonetheless showed differences in prognosis. Known gene copy number variations and mutations in key cancer-associated genes differed between subtypes, but the subtypes provided molecular information beyond that contained in these variables. Morphological features significantly differed between subtypes. The objective existence of the subtypes and their clinical and molecular characteristics were validated in an independent set of 720 CRC expression profiles. Our subtypes provide a novel perspective on the heterogeneity of CRC. The proposed subtypes should be further explored retrospectively on existing clinical trial datasets and, when sufficiently robust, be prospectively assessed for clinical relevance in terms of prognosis and treatment response predictive capacity. Original microarray data were uploaded to the ArrayExpress database (http

  7. MILLIMETER-SCALE GENETIC GRADIENTS AND COMMUNITY-LEVEL MOLECULAR CONVERGENCE IN A HYPERSALINE MICROBIAL MAT

    Energy Technology Data Exchange (ETDEWEB)

    Fenner, Marsha W; Kunin, Victor; Raes, Jeroen; Harris, J. Kirk; Spear, John R.; Walker, Jeffrey J.; Ivanova, Natalia; Mering, Christian von; Bebout, Brad M.; Pace, Norman R.; Bork, Peer; Hugenholtz, Philip

    2008-04-30

    To investigate the extent of genetic stratification in structured microbial communities, we compared the metagenomes of 10 successive layers of a phylogenetically complex hypersaline mat from Guerrero Negro, Mexico. We found pronounced millimeter-scale genetic gradients that are consistent with the physicochemical profile of the mat. Despite these gradients, all layers displayed near identical and acid-shifted isoelectric point profiles due to a molecular convergence of amino acid usage indicating that hypersalinity enforces an overriding selective pressure on the mat community.

  8. Tuning molecular level alignment and work function modification through self-assembled monolayers on noble metals: theoretical perspectives

    International Nuclear Information System (INIS)

    Full text: There is currently significant interest in highly-ordered, self-assembled monolayers (SAMs) on (noble) metal surfaces, inspired both by the emergence of the field of molecular electronics alongside the high potential for SAMs to improve the properties of more conventional device structures. SAMs are also used to control surface reactivity and for chemical sensing applications. In order to tune the interface properties and to endow the self-assembled systems with functionality suitable for use in either macroscopic or nanoscale devices, the use of π-conjugated systems is highly promising and the focus of intense, multidisciplinary research. The goal of the present study is to provide an in-depth description of the electronic structure of the interface between metallic substrates and covalently bound conjugated molecules. In this way, we expect to devise strategies to tune the interaction and thus the properties of the investigated systems and eventually to gain a full understanding of the processes governing the electronics of metal/organic interfaces. Here, we describe a first step in that direction: we study conjugated SAMs consisting of molecules with widely varied molecular ionization potentials, different conjugated backbones with different polarizabilities, and monolayers with varying degrees of coverage. We consider noble metals with varying work functions such as Au, Ag, and Pt, different molecule docking groups and investigate the effects of mechanical stress on the organic system. Using DFT band-structure-type methods, the details of the interface morphology, charge transfer between the metal and the molecules, interface dipoles, molecular layer depolarization, and work function modifications as well as the alignment between metallic and molecular levels are described. Our thorough analysis provides results that are sometimes a priori unexpected, like the finding that by properly tuning the molecular structure, the level alignment between the

  9. Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity.

    Science.gov (United States)

    Chiu, Isaac M; Barrett, Lee B; Williams, Erika K; Strochlic, David E; Lee, Seungkyu; Weyer, Andy D; Lou, Shan; Bryman, Gregory S; Roberson, David P; Ghasemlou, Nader; Piccoli, Cara; Ahat, Ezgi; Wang, Victor; Cobos, Enrique J; Stucky, Cheryl L; Ma, Qiufu; Liberles, Stephen D; Woolf, Clifford J

    2014-01-01

    The somatosensory nervous system is critical for the organism's ability to respond to mechanical, thermal, and nociceptive stimuli. Somatosensory neurons are functionally and anatomically diverse but their molecular profiles are not well-defined. Here, we used transcriptional profiling to analyze the detailed molecular signatures of dorsal root ganglion (DRG) sensory neurons. We used two mouse reporter lines and surface IB4 labeling to purify three major non-overlapping classes of neurons: 1) IB4(+)SNS-Cre/TdTomato(+), 2) IB4(-)SNS-Cre/TdTomato(+), and 3) Parv-Cre/TdTomato(+) cells, encompassing the majority of nociceptive, pruriceptive, and proprioceptive neurons. These neurons displayed distinct expression patterns of ion channels, transcription factors, and GPCRs. Highly parallel qRT-PCR analysis of 334 single neurons selected by membership of the three populations demonstrated further diversity, with unbiased clustering analysis identifying six distinct subgroups. These data significantly increase our knowledge of the molecular identities of known DRG populations and uncover potentially novel subsets, revealing the complexity and diversity of those neurons underlying somatosensation. PMID:25525749

  10. Global plant-responding mechanisms to salt stress: physiological and molecular levels and implications in biotechnology.

    Science.gov (United States)

    Tang, Xiaoli; Mu, Xingmin; Shao, Hongbo; Wang, Hongyan; Brestic, Marian

    2015-01-01

    The increasing seriousness of salinization aggravates the food, population and environmental issues. Ameliorating the salt-resistance of plants especially the crops is the most effective measure to solve the worldwide problem. The salinity can cause damage to plants mainly from two aspects: hyperosmotic and hyperionic stresses leading to the restrain of growth and photosynthesis. To the adverse effects, the plants derive corresponding strategies including: ion regulation and compartmentalization, biosynthesis of compatible solutes, induction of antioxidant enzymes and plant hormones. With the development of molecular biology, our understanding of the molecular and physiology knowledge is becoming clearness. The complex signal transduction underlying the salt resistance is being illuminated brighter and clearer. The SOS pathway is the central of the cell signaling in salt stress. The accumulation of the compatible solutes and the activation of the antioxidant system are the effective measures for plants to enhance the salt resistance. How to make full use of our understanding to improve the output of crops is a huge challenge for us, yet the application of the genetic engineering makes this possible. In this review, we will discuss the influence of the salt stress and the response of the plants in detail expecting to provide a particular account for the plant resistance in molecular, physiological and transgenic fields. PMID:24738851

  11. Molecular-Level Insights into Photocatalysis from Scanning Probe Microscopy Studies on TiO2(110)

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, Michael A.; Lyubinetsky, Igor

    2013-06-12

    The field of heterogeneous photocatalysis has grown considerably in the decades since Fujishima and Honda's ground-breaking publications of photoelectrochemistry on TiO2. Numerous review articles continue to point to both progress made in the use of heterogeneous materials (such as TiO2) to perform photoconversion processes, and the many opportunities and challenges in heterogeneous photocatalysis research such as solar energy conversion and environmental remediation. The past decade has also seen an increase in the use of molecular-level approaches applied to model single crystal surfaces in an effort to obtain new insights into photocatalytic phenomena. In particular, scanning probe techniques (SPM) have enabled researchers to take a ‘nanoscale’ approach to photocatalysis that includes interrogation of the reactivities of specific sites and adsorbates on a model photocatalyst surface. The rutile TiO2(110) surface has become the prototypical oxide single crystal surface for fundamental studies of many interfacial phenomena. In particular, TiO2(110) has become an excellent model surface for probing photochemical and photocatalytic reactions at the molecular level. A variety of experimental approaches have emerged as being ideally suited for studying photochemical reactions on TiO2(110), including desorption-oriented approaches and electronic spectroscopies, but perhaps the most promising techniques for evaluating site-specific properties are those of SPM. In this review, we highlight the growing use of SPM techniques in providing molecular-level insights into surface photochemistry on the model photocatalyst surface of rutile TiO2(110). Our objective is to both illustrate the unique knowledge that scanning probe techniques have already provided the field of photocatalysis, and also to motivate a new generation of effort into the use of such approaches to obtain new insights into the molecular level details of photochemical events occurring at interfaces

  12. Protective effects of anti-ricin A-chain RNA aptamer against ricin toxicity

    Institute of Scientific and Technical Information of China (English)

    Shaoan Fan; Feng Wu; Frank Martiniuk; Martha L Hale; Andrew D Ellington; Kam-Meng Tchou-Wong

    2008-01-01

    AIM: To investigate the therapeutic potential of an RNA ligand (aptamer) specific for the catalytic ricin A-chain (RTA), the protective effects of a 31-nucleo-tide RNA aptamer (31RA), which formed a high affinity complex with RTA, against ricin-induced toxicity in cell-based luciferase translation and ceil cytotoxicity assays were evaluated. METHODS: To test the therapeutic potential of anti-RTA aptamers in Chinese hamster ovary (CHO) AA8 cells stably transfected with a tetracycline regulato able promoter, ricin ribotoxicity was measured us-ing luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tet-razolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. RESULTS: Inhibition of protein synthesis by ricin in CHO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against degly-cosylated RTA (dgA) neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation as-say. 31RA aptamer also partially neutralized the inhibi-tory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CHO AA8 cells. CONCLUSION- We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in ceil-based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity rep-resents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication.

  13. Multi-study integration of brain cancer transcriptomes reveals organ-level molecular signatures.

    Directory of Open Access Journals (Sweden)

    Jaeyun Sung

    Full Text Available We utilized abundant transcriptomic data for the primary classes of brain cancers to study the feasibility of separating all of these diseases simultaneously based on molecular data alone. These signatures were based on a new method reported herein--Identification of Structured Signatures and Classifiers (ISSAC--that resulted in a brain cancer marker panel of 44 unique genes. Many of these genes have established relevance to the brain cancers examined herein, with others having known roles in cancer biology. Analyses on large-scale data from multiple sources must deal with significant challenges associated with heterogeneity between different published studies, for it was observed that the variation among individual studies often had a larger effect on the transcriptome than did phenotype differences, as is typical. For this reason, we restricted ourselves to studying only cases where we had at least two independent studies performed for each phenotype, and also reprocessed all the raw data from the studies using a unified pre-processing pipeline. We found that learning signatures across multiple datasets greatly enhanced reproducibility and accuracy in predictive performance on truly independent validation sets, even when keeping the size of the training set the same. This was most likely due to the meta-signature encompassing more of the heterogeneity across different sources and conditions, while amplifying signal from the repeated global characteristics of the phenotype. When molecular signatures of brain cancers were constructed from all currently available microarray data, 90% phenotype prediction accuracy, or the accuracy of identifying a particular brain cancer from the background of all phenotypes, was found. Looking forward, we discuss our approach in the context of the eventual development of organ-specific molecular signatures from peripheral fluids such as the blood.

  14. Higher-level molecular phylogeny of the water mites (Acariformes: Prostigmata: Parasitengonina: Hydrachnidiae).

    Science.gov (United States)

    Dabert, Miroslawa; Proctor, Heather; Dabert, Jacek

    2016-08-01

    With nearly 6000 named species, water mites (Hydrachnidiae) represent the largest group of arachnids to have invaded and extensively diversified in freshwater habitats. Water mites together with three other lineages (the terrestrial Erythraiae and Trombidiae, and aquatic Stygothrombiae), make up the hyporder Parasitengonina, which is characterized by having parasitic larvae and predatory nymphs and adults. Relationships between the Hydrachnidiae and other members of the Parasitengonina are unclear, as are relationships among the major lineages of water mites. Monophyly of water mites has been asserted, with the possible exception of the morphologically distinctive Hydrovolzioidea. Here we infer the phylogeny of water mites using multiple molecular markers and including representatives of all superfamilies of Hydrachnidiae and of almost all other Parasitengonina. Our results support a monophyletic Parasitengonina including Trombidiae, Stygothrombiae, and Hydrachnidiae. A monophyletic Hydrachnidiae, including Hydrovolzioidea, is strongly supported. Terrestrial Parasitengonina do not form a monophyletic sister group to water mites. Stygothrombiae is close to water mites but is not nested within this clade. Water mites appear to be derived from ancestors close to Stygothrombiae or the erythraoid group Calyptostomatoidea; however, this relationship is not clear because of extremely short branches in this part of the parasitengonine tree. We recovered with strong support all commonly accepted water mite superfamilies except for Hydryphantoidea, which is clearly paraphyletic. Our data support the previously proposed clades Protohydrachnidia (Hydrovolzioidea and Eylaoidea), Euhydrachnidia (all remaining superfamilies), and the euhydrachnid subclade Neohydrachnidia (Lebertioidea, Hydrachnoidea, Hygrobatoidea, and Arrenuroidea). We found that larval leg structure and locomotory behavior are strongly congruent with the molecular phylogeny. Other morphological and behavioral

  15. Molecular Phylogeny of the Astrophorida (Porifera, Demospongiaep) Reveals an Unexpected High Level of Spicule Homoplasy

    Science.gov (United States)

    Cárdenas, Paco; Xavier, Joana R.; Reveillaud, Julie; Schander, Christoffer; Rapp, Hans Tore

    2011-01-01

    Background The Astrophorida (Porifera, Demospongiaep) is geographically and bathymetrically widely distributed. Systema Porifera currently includes five families in this order: Ancorinidae, Calthropellidae, Geodiidae, Pachastrellidae and Thrombidae. To date, molecular phylogenetic studies including Astrophorida species are scarce and offer limited sampling. Phylogenetic relationships within this order are therefore for the most part unknown and hypotheses based on morphology largely untested. Astrophorida taxa have very diverse spicule sets that make them a model of choice to investigate spicule evolution. Methodology/Principal Findings With a sampling of 153 specimens (9 families, 29 genera, 89 species) covering the deep- and shallow-waters worldwide, this work presents the first comprehensive molecular phylogeny of the Astrophorida, using a cytochrome c oxidase subunit I (COI) gene partial sequence and the 5′ end terminal part of the 28S rDNA gene (C1-D2 domains). The resulting tree suggested that i) the Astrophorida included some lithistid families and some Alectonidae species, ii) the sub-orders Euastrophorida and Streptosclerophorida were both polyphyletic, iii) the Geodiidae, the Ancorinidae and the Pachastrellidae were not monophyletic, iv) the Calthropellidae was part of the Geodiidae clade (Calthropella at least), and finally that v) many genera were polyphyletic (Ecionemia, Erylus, Poecillastra, Penares, Rhabdastrella, Stelletta and Vulcanella). Conclusion The Astrophorida is a larger order than previously considered, comprising ca. 820 species. Based on these results, we propose new classifications for the Astrophorida using both the classical rank-based nomenclature (i.e., Linnaean classification) and the phylogenetic nomenclature following the PhyloCode, independent of taxonomic rank. A key to the Astrophorida families, sub-families and genera incertae sedis is also included. Incongruences between our molecular tree and the current classification can

  16. Molecular responses during cadmium-induced stress in Daphnia magna: Integration of differential gene expression with higher-level effects

    International Nuclear Information System (INIS)

    DNA microarrays offer great potential in revealing insight into mechanistic toxicity of contaminants. The aim of the present study was (i) to gain insight in concentration- and time-dependent cadmium-induced molecular responses by using a customized Daphnia magna microarray, and (ii) to compare the gene expression profiles with effects at higher levels of biological organization (e.g. total energy budget and growth). Daphnids were exposed to three cadmium concentrations (nominal value of 10, 50, 100 μg/l) for two time intervals (48 and 96 h). In general, dynamic expression patterns were obtained with a clear increase of gene expression changes at higher concentrations and longer exposure duration. Microarray analysis revealed cadmium affected molecular pathways associated with processes such as digestion, oxygen transport, cuticula metabolism and embryo development. These effects were compared with higher-level effects (energy budgets and growth). For instance, next to reduced energy budgets due to a decline in lipid, carbohydrate and protein content, we found an up-regulated expression of genes related to digestive processes (e.g. α-esterase, cellulase, α-amylase). Furthermore, cadmium affected the expression of genes coding for proteins involved in molecular pathways associated with immune response, stress response, cell adhesion, visual perception and signal transduction in the present study

  17. Selected materials of the international workshop on radiation risk and its origin at molecular and cellular level

    International Nuclear Information System (INIS)

    The workshop ''International Workshop on Radiation Risk and its Origin at Molecular and Cellular Level'' was held at The Tokai Research Establishment, Japan Atomic Energy Research Institute, on the 6th and 7th of February 2003. The Laboratory of Radiation Risk Analysis of JAERI organized it. This international workshop attracted scientists from several different scientific areas, including radiation physics, radiation biology, molecular biology, crystallography of biomolecules, modeling and bio-informatics. Several foreign and domestic keynote speakers addresses the very fundamental areas of radiation risk and tried to establish a link between the fundamental studies at the molecular and cellular level and radiation damages at the organism. The symposium consisted of 13 oral lectures, 10 poster presentations and panel discussion. The 108 participants attended the workshop. This publication comprises of proceedings of oral and poster presentations where available. For the rest of contributions the abstracts or/and selections of presentation materials are shown instead. The 5 papers are indexed individually. (J.P.N.)

  18. Dynamics of cooperative emissions in a cascade three-level molecular system driven by an ultrashort laser pulse

    International Nuclear Information System (INIS)

    This paper investigates the dynamics of cooperative emissions in a cascade three-level system driven by an ultrashort laser pulse by solving numerically the full-wave Maxwell–Bloch equations. The 4, 4'-bis(dimethylamino) stilbene molecule is used as the model molecule because of its strong two-photon absorption property. The two-colour cooperative emissions are studied as functions of molecular number density and dephasing rate of the dipole coherence. The propagation effects on the evolution of the cooperative radiations are also taken into account. The cooperative radiations are enhanced for large number density of the molecule, while the fast dephasing of the dipole coherence reduces the intensity of the cooperative radiations and delays the emission times or even inhibits the formation of the emissions. The delay time of the radiation decreases with the increase of the molecular number density and the propagation distance. (classical areas of phenomenology)

  19. Study on the toxic interaction of methanol, ethanol and propanol against the bovine hemoglobin (BHb) on molecular level

    Science.gov (United States)

    Jun, Chai; Xue, Yan; Liu, Rutao; Wang, Meijie

    2011-09-01

    The toxic interaction of methanol, ethanol and propanol with bovine hemoglobin (BHb) at protein molecular level was studied by resonance light scattering (RLS), fluorescence, ultraviolet-visible absorption (UV-vis) and circular dichroism (CD) techniques. The experimental results showed that the three alcohols all had toxic effects on BHb and the effects increased along with the increasing alcohol dose. The results of RLS and fluorescence spectroscopy showed that alcohols can denature BHb. They changed the microenvironment of amino acid residues and led to molecular aggregation. The decreasing order of the influence is propanol, ethanol and methanol. The results of UV-vis and CD spectra revealed that alcohols led to conformational changes of BHb, including the loosening of the skeleton structure and the decreasing of α-helix in the second structure. The changes generated by propanol were much larger than those by methanol and ethanol.

  20. Coexistence of spinodal instability and thermal nucleation in thin-film rupture:Insights from molecular levels

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Trung D [ORNL; Fuentes-Cabrera, Miguel A [ORNL; Fowlkes, Jason Davidson [ORNL; Rack, Philip D [ORNL

    2014-01-01

    Despite extensive investigation using hydrodynamic models and experiments over the past decades, there remain open questions regarding the origin of the initial rupture of thin liquid films. One of the reasons that makes it difficult to identify the rupture origin is the coexistence of two dewettingmechanisms, namely, thermal nucleation and spinodal instability, as observed in many experimental studies. Using a coarse-grained model and large-scale molecular dynamics simulations, we are able to characterize the very early stage of dewetting in nanometer-thick liquid-metal films wetting a solid substrate. We observe the features characteristic of both spinodal instability and thermal nucleation in the spontaneously dewetting films and show that these two macroscopic mechanisms share a common origin at molecular levels.