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Artykuły w czasopismach na temat "Thrombin binding aptamer"
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Ponzo, Irene, Friederike M. Möller, Herwin Daub i Nena Matscheko. "A DNA-Based Biosensor Assay for the Kinetic Characterization of Ion-Dependent Aptamer Folding and Protein Binding". Molecules 24, nr 16 (8.08.2019): 2877. http://dx.doi.org/10.3390/molecules24162877.
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Therapeutic and diagnostic nucleic acid aptamers are designed to bind tightly and specifically to their target. The combination of structural and kinetic analyses of aptamer interactions has gained increasing importance. Here, we present a fluorescence-based switchSENSE aptasensor for the detailed kinetic characterization of aptamer–analyte interaction and aptamer folding, employing the thrombin-binding aptamer (TBA) as a model system. Thrombin-binding aptamer folding into a G-quadruplex and its binding to thrombin strongly depend on the type and concentration of ions present in solution. We observed conformational changes induced by cations in real-time and determined the folding and unfolding kinetics of the aptamer. The aptamer’s affinity for K+ was found to be more than one order of magnitude higher than for other cations (K+ > NH4+ >> Na+ > Li+). The aptamer’s affinity to its protein target thrombin in the presence of different cations followed the same trend but differed by more than three orders of magnitude (KD = 0.15 nM to 250 nM). While the stability (kOFF) of the thrombin–TBA complex was similar in all conditions, the cation type strongly influenced the association rate (kON). These results demonstrated that protein–aptamer binding is intrinsically related to the correct aptamer fold and, hence, to the presence of stabilizing ions. Because fast binding kinetics with on-rates exceeding 108 M−1s−1 can be quantified, and folding-related phenomena can be directly resolved, switchSENSE is a useful analytical tool for in-depth characterization of aptamer–ion and aptamer–protein interactions.
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Kim, Jieun, Dajeong Kim i Jong Bum Lee. "DNA aptamer-based carrier for loading proteins and enhancing the enzymatic activity". RSC Advances 7, nr 3 (2017): 1643–45. http://dx.doi.org/10.1039/c6ra25507h.
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Here, we synthesized DNA microparticles comprised of thrombin binding aptamers via rolling circle amplification (RCA). These DNA aptamer particles could successfully load a number of thrombins and the complexes have shown improved thrombin activity.
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Poturnayová, Alexandra, Maja Šnejdárková i Tibor Hianik. "DNA aptamer configuration affects the sensitivity and binding kinetics of thrombin". Acta Chimica Slovaca 5, nr 1 (1.04.2012): 53–58. http://dx.doi.org/10.2478/v10188-012-0009-z.
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DNA aptamer configuration affects the sensitivity and binding kinetics of thrombinThrombin is serine protease involved in the coagulation cascade, which converts soluble fibrinogen into insoluble strands of fibrin - a matrix of the blood clot formation. Development of the sensitive method of the thrombin detection in nanomolar level is important for clinical practice. In this work we applied acoustic thickness shear mode method (TSM) for study the binding of human thrombin depending on DNA aptamer configuration. We compared sensitivity of detection and binding kinetics of the thrombin to the conventional DNA aptamers and aptamer dimers immobilized at the surface of quartz crystal transducer. We have shown that aptasensors based on aptamer dimers more sensitively detect thrombin. The aptamer-thrombin complexes were also more stable as revealed from equilibrium dissociation constant,KD, that was 4 times lower for aptamer dimers in comparison with conventional aptamers. Determination of motional resistance,Rm, from acoustic impedance analysis allowed us to find important differences in physico-chemical properties of layers formed by conventional aptamers and aptamer dimers.
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Zhdanov, Gleb, Alexander Arutyunyuan, Alexey Kopylov i Elena Zavyalova. "Energy Dissipation Hypothesis Applied to Enhance the Affinity of Thrombin Binding Aptamer". Biophysica 1, nr 2 (14.05.2021): 179–93. http://dx.doi.org/10.3390/biophysica1020014.
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Nucleic acid aptamers are artificial recognizing molecules that are capable of specific binding to a wide variety of targets. Aptamers are commonly selected from a huge library of oligonucleotides and improved by introducing several mutations or modular constructions. Although aptamers hold great promise as therapeutic and diagnostic tools, no simple approach to improve their affinity has been suggested yet. Our recent analysis of aptamer–protein complexes revealed that aptamer affinity correlates with the size of an amino acid sidechain in the protein interface that was explained by efficient dissipation of the energy released during complex formation. G-quadruplex-based thrombin aptamers are not involved in the described dependence. Moreover, aptamers to the same thrombin site have 100-fold differences in affinity. Here we focused on a detailed analysis of the nucleic acid interface of thrombin–aptamer complexes. High affinity of the aptamers was shown to correlate with the solvent accessibility of the apolar part of recognizing loops. To prove the concept experimentally, these loops were modified to enhance contact with the solvent. Dissociation rates of the aptamer–thrombin complexes were drastically slowed due to these modifications. In full correspondence with the energy dissipation hypothesis, the modifications improved both the stability of the G-quadruplexes and affinity to thrombin. The most evident effect was shown for unstable Na+-coordinated G-quadruplexes. These data are of high interest for a directed improvement of aptamers introducing unnatural modifications into the ‘hot spot’ residues.
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Beyer, Stefan, Wendy U. Dittmer, Andreas Reuter i Friedrich C. Simmel. "Controlled Release of Thrombin Using Aptamer-Based Nanodevices". Advances in Science and Technology 53 (październik 2006): 116–21. http://dx.doi.org/10.4028/www.scientific.net/ast.53.116.
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Aptamers are DNA or RNA single strands that have been selected from random pools based on their ability to bind ligands. Like antibodies, aptamers are highly specific to their targets, and thus have many potential uses in biomedicine and biotechnology. We report here on the construction of a protein-binding molecular device based on a DNA aptamer, which can be instructed to hold or release the human blood-clotting factor, α-thrombin, depending on an operator DNA sequence addressing it. In the operation of this DNA nanodevice, the thrombin-binding DNA aptamer is switched between a binding and a non-binding form. This is achieved by sequentially hybridizing and removing a DNA single strand to the protein binding region of the aptamer. This principle of operation is limited as the switching sequence is determined by the protein-binding sequence. To overcome this limitation we introduce a DNA signal translation device that allows the operation of aptamers with arbitrary sequences. The function of the translator is based on branch migration and the action of the endonuclease FokI. The modular design of the translator facilitates the adaptation of the device to various input or output sequences.
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Kolganova, Natalia A., Vladimir B. Tsvetkov, Andrey A. Stomakhin, Sergei A. Surzhikov, Edward N. Timofeev i Irina V. Varizhuk. "Alpha-Deoxyguanosine to Reshape the Alpha-Thrombin Binding Aptamer". International Journal of Molecular Sciences 24, nr 9 (7.05.2023): 8406. http://dx.doi.org/10.3390/ijms24098406.
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Modification of DNA aptamers is aimed at increasing their thermodynamic stability, and improving affinity and resistance to biodegradation. G-quadruplex DNA aptamers are a family of affinity ligands that form non-canonical DNA assemblies based on a G-tetrads stack. Modification of the quadruplex core is challenging since it can cause complete loss of affinity of the aptamer. On the other hand, increased thermodynamic stability could be a worthy reward. In the current paper, we developed new three- and four-layer modified analogues of the thrombin binding aptamer with high thermal stability, which retain anticoagulant activity against alpha-thrombin. In the modified aptamers, one or two G-tetrads contained non-natural anti-preferred alpha-deoxyguanosines at specific positions. The use of this nucleotide analogue made it possible to control the topology of the modified structures. Due to the presence of non-natural tetrads, we observed some decrease in the anticoagulant activity of the modified aptamers compared to the natural prototype. This negative effect was completely compensated by conjugation of the aptamers with optimized tripeptide sequences.
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Funck, Timon, Tim Liedl i Wooli Bae. "Dual Aptamer-Functionalized 3D Plasmonic Metamolecule for Thrombin Sensing". Applied Sciences 9, nr 15 (26.07.2019): 3006. http://dx.doi.org/10.3390/app9153006.
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DNA nanotechnology offers the possibility to rationally design structures with emergent properties by precisely controlling their geometry and functionality. Here, we demonstrate a DNA-based plasmonic metamolecule that is capable of sensing human thrombin proteins. The chiral reconfigurability of a DNA origami structure carrying two gold nanorods was used to provide optical read-out of thrombin binding through changes in the displayed plasmonic circular dichroism. In our experiments, each arm of the structure was modified with one of two different thrombin-binding aptamers—thrombin-binding aptamer (TBA) and HD22—in such a way that a thrombin molecule could be sandwiched by the aptamers to lock the metamolecule in a state of defined chirality. Our structure exhibited a Kd of 1.4 nM, which was an order of magnitude lower than those of the individual aptamers. The increased sensitivity arose from the avidity gained by the cooperative binding of the two aptamers, which was also reflected by a Hill coefficient of 1.3 ± 0.3. As we further exploited the strong plasmonic circular dichroism (CD) signals of the metamolecule, our method allowed one-step, high sensitivity optical detection of human thrombin proteins in solution.
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Seelam Prabhakar, Preethi, Richard A. Manderville i Stacey D. Wetmore. "Impact of the Position of the Chemically Modified 5-Furyl-2′-Deoxyuridine Nucleoside on the Thrombin DNA Aptamer–Protein Complex: Structural Insights into Aptamer Response from MD Simulations". Molecules 24, nr 16 (10.08.2019): 2908. http://dx.doi.org/10.3390/molecules24162908.
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Aptamers are functional nucleic acids that bind to a range of targets (small molecules, proteins or cells) with a high affinity and specificity. Chemically-modified aptamers are of interest because the incorporation of novel nucleobase components can enhance aptamer binding to target proteins, while fluorescent base analogues permit the design of functional aptasensors that signal target binding. However, since optimally modified nucleoside designs have yet to be identified, information about how to fine tune aptamer stability and target binding affinity is required. The present work uses molecular dynamics (MD) simulations to investigate modifications to the prototypical thrombin-binding aptamer (TBA), which is a 15-mer DNA sequence that folds into a G-quadruplex structure connected by two TT loops and one TGT loop. Specifically, we modeled a previously synthesized thymine (T) analog, namely 5-furyl-2′-deoxyuridine (5FurU), into each of the six aptamer locations occupied by a thymine base in the TT or TGT loops of unbound and thrombin bound TBA. This modification and aptamer combination were chosen as a proof-of-principle because previous experimental studies have shown that TBA displays emissive sensitivity to target binding based on the local environment polarity at different 5FurU modification sites. Our simulations reveal that the chemically-modified base imparts noticeable structural changes to the aptamer without affecting the global conformation. Depending on the modification site, 5FurU performance is altered due to changes in the local environment, including the modification site structural dynamics, degree of solvent exposure, stacking with neighboring bases, and interactions with thrombin. Most importantly, these changes directly correlate with the experimentally-observed differences in the stability, binding affinity and emissive response of the modified aptamers. Therefore, the computational protocols implemented in the present work can be used in subsequent studies in a predictive way to aid the fine tuning of aptamer target recognition for use as biosensors (aptasensors) and/or therapeutics.
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Zeng, Xinling, Qing Zhou, Liyan Wang, Xiaoxian Zhu, Kuiyan Cui, Xinsheng Peng, Terry W. J. Steele, Huizhi Chen, Hui Xu i Yubin Zhou. "A Fluorescence Kinetic-Based Aptasensor Employing Stilbene Isomerization for Detection of Thrombin". Materials 14, nr 22 (16.11.2021): 6927. http://dx.doi.org/10.3390/ma14226927.
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It is important to detect thrombin due to its physiological and pathological roles, where rapid and simple analytical approaches are needed. In this study, an aptasensor based on fluorescence attenuation kinetics for the detection of thrombin is presented, which incorporates the features of stilbene and aptamer. We designed and synthesized an aptasensor by one-step coupling of stilbene compound and aptamer, which employed the adaptive binding of the aptamer with thrombin to cause a change in stilbene fluorescence attenuation kinetics. The sensor realized detection of thrombin by monitoring the variation in apparent fluorescence attenuation rate constant (kapp), which could be further used for probing of enzyme–aptamer binding. In comprehensive studies, the developed aptasensor presented satisfactory performance on repeatability, specificity, and regeneration capacity, which realized rapid sensing (10 s) with a limit of detection (LOD) of 0.205 μM. The strategy was successful across seven variants of thrombin aptasensors, with tunable kapp depending on the SITS (4-Acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid disodium salt hydrate) grafting site. Analyte detection mode was demonstrated in diluted serum, requiring no separation or washing steps. The new sensing mode for thrombin detection paves a way for high-throughput kinetic-based sensors for exploiting aptamers targeted at clinically relevant proteins.
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Russo Krauss, Irene, Andrea Pica, Antonello Merlino, Lelio Mazzarella i Filomena Sica. "Duplex–quadruplex motifs in a peculiar structural organization cooperatively contribute to thrombin binding of a DNA aptamer". Acta Crystallographica Section D Biological Crystallography 69, nr 12 (19.11.2013): 2403–11. http://dx.doi.org/10.1107/s0907444913022269.
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Potent second-generation thrombin aptamers adopt a duplex–quadruplex bimodular folding and recognize thrombin exosite II with very high affinity and specificity. A sound model of these oligonucleotides, either free or in complex with thrombin, is not yet available. Here, a structural study of one of these aptamers, HD22-27mer, is presented. The crystal structure of this aptamer in complex with thrombin displays a novel architecture in which the helical stem is enchained to a pseudo-G-quadruplex. The results also underline the role of the residues that join the duplex and quadruplex motifs and control their recruitment in thrombin binding.
Rozprawy doktorskie na temat "Thrombin binding aptamer"
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Trapaidze, Ana. "Integration of thrombin-binding aptamers in point-of-care devices for continuous monitoring of thrombin in plasma". Thesis, Toulouse 3, 2015. http://www.theses.fr/2015TOU30050/document.
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La thrombine est l'enzyme principale dans le processus d'hémostase. Les dérèglements de la concentration de thrombine clinique prédisposent les patients à des complications hémorragiques ou thromboemboliques. Le suivi en temps réel de la thrombine dans le sang est donc nécessaire pour améliorer le traitement de patients en état critique. Les aptamères, qui sont de courts nucléotides monobrins semblent constituer des candidats prometteurs pour la reconnaissance moléculaire dans les biocapteurs. L'objectif de ces travaux est l'étude de différentes solutions d'intégration des aptamères dans des dispositifs de diagnostic de type "point of care" pour le suivi en continu de la thrombine dans le plasma. La cinétique d'interaction des aptamères avec la thrombine et leur spécificité vis-à-vis de la prothrombine et des inhibiteurs de la thrombine ont été étudiés par résonance par plasmons de surface. Ces travaux ont démontré la faible spécificité de l'aptamère HD1 vis-à-vis de la thrombine, et la présence d'interactions non-spécifiques avec la prothrombine, les inhibiteurs naturels de la thrombine et l'albumine. Inversement, nous avons observé une bonne affinité de l'aptamère HD22 avec la même liste de cible. Parallèlement, nous avons évalué des stratégies d'intégration d'aptamères dans des dispositifs d'analyse. Le principe de reconnaissance a ensuite été validé et la possibilité de détecter la thrombine dans des gammes de concentration de 5 à 500nM a été démontrée. Enfin, afin d'augmenter la spécificité de la détection de la thrombine, nous avons proposé une nouvelle approche basée sur l'ingénierie de structures dimères interconnectant HD1 et HD22Thrombin is the central enzyme in the process of hemostasis. Normally, in vivo concentration of thrombin is rigorously regulated; however, clinically impaired or unregulated thrombin generation predisposes patients either to hemorrhagic or thromboembolic complications. Monitoring thrombin in real-time is therefore needed to enable rapid and accurate determination of drug administration strategy for patients under vital threat. Aptamers, short single-stranded oligonucleotide ligands represent promising candidates as biorecognition elements for new-generation biosensors. The aim of this PhD work therefore is to investigate different solutions for the integration of thrombin-binding aptamers in point-of-care devices for continuous monitoring of thrombin in plasma. The kinetics of aptamer interaction with thrombin and specificity towards prothrombin and thrombin - inhibitor complexes was rigorously investigated using Surface Plasmon Resonance. These experiments unveiled the complex character of interaction of the HD1 with thrombin, confirming nonspecific interactions with prothrombin, natural inhibitors of thrombin, serum albumin whereas another 29-bp aptamer HD22 proved to be highly affine and specific towards thrombin. On the other hand we explored aptamer integration options. We validated the principle and at the same managed to detect different concentrations of thrombin (5-500 nM). We finally proposed a novel approach to increase sensitivity and specificity for thrombin detection based on the engineering of aptadimer structures bearing aptamers HD1and HD22 interconnected with a nucleic acid spacer
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Pal, Sohini. "Nanopore Based Single-molecule Sensors". Thesis, 2020. https://etd.iisc.ac.in/handle/2005/5457.
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In the past two decades nanopores have been used as highly sensitive
detection systems for exploring the properties of analytes at single molecule
resolution. The small dimensions of a nanopore permit the molecule of interest to
be confined within it, allowing for the extraction of valuable information relating to
its physical and chemical properties. Single molecule analysis, as opposed to bulk
measurements does not involve ensemble averaging. Hence, short-lived states
such as an intermediate configuration during a conformational change can be
observed directly, while such states would be masked in the bulk assay.
The main project described in this thesis involves the design and fabrication
of a hybrid silicon nitride-DNA origami nanopore system for use in biosensing of
proteins. We used the nanopore system to experimentally observe the effect of
forces between the translocating molecule and nanopore with a focus on the
electro kinetics inside the pore and escape rate problem. These are further verified
by finite element simulations and MATLAB simulations which enables us to
investigate the physics behind the different types of events that we observe.
The key findings from this work can be summarized as follows. We report
on an operating regime of this nanopore sensor, characterized by attractive
interactions between the nanoparticle and the pore, where the dwell time is
exponentially sensitive to the target-pore interaction. We used negatively and
positively charged gold nanoparticles to control the strength of their interaction with
the negatively charged silicon nitride pore. Our experiments revealed how this
modulation of the electrostatic force greatly affects the ionic current with an
exponential dependance of dwell times. A stochastic model is developed for
analyzing this analyte-pore interaction based on the well-known Kramer’s problem
of escape from a barrier.Finally, the nitride nanopore was functionalized using DNA
origami with thrombin binding aptamer (TBA15), a well studied 15-mer aptamer
DNA sequence that binds selectively with thrombin protein. Consistent with our
previous experiment, we observed current traces with large dwell time blockades
for thrombin whereas for another protein the trace contained minimal dwell time current enhancements. The presence of TBA15 aptamer increased the interaction
energy between the thrombin and the nanopore resulting in a blockage with
comparatively larger dwell time and enabled us in sensing thrombin at
concentrations as low as 20nM.
Nanopore technology will remain an important field of science in the
21st century. We believe equipped with our understanding of nanopore analysis, in
future we will be able to detect and unravel important physical phenomena in the
single molecule world.
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chen, Ren-hao, i 陳人豪. "Studies of the Binding Mechanism between Aptamers and Thrombin by Surface Plasmon Resonance and Circular Dichroism". Thesis, 2010. http://ndltd.ncl.edu.tw/handle/09067407226002566458.
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碩士國立中央大學
化學工程與材料工程研究所
98
This study characterizes the interaction mechanism between aptamers and human thrombin by surface plasmon resonance (SPR) and ciucular dichroism (CD). Thrombin, a multifunctional serine protease, has both procoagulant and anticoagulant functions in human blood. Thrombin involves two electropositive exosites；one is fibrinogen-binding site and the other is heparin-binding site. Two thrombin-binding aptamers have been selected by SELEX technique over the past decade, respectively. One is 15-mer aptamer binds at fibrinogen-binding site of thrombin, while 29-mer aptamer binds at the heparin binding site of thrombin. In the past years many papers have reported the interaction between 15-mer aptamer and thrombin, however the difference of the two aptamers bind to thrombin is still lacking and worth of investigation. In this study, we combined kinetics and conformational information to compare the binding mechanism between these two aptamers with thrombin. Two experiments were mainly performed in this investigation. CD assay demonstrated the comformational feature of different aptamers binding to thrombin, while SPR provided kinetic constant (Ka) in different binding parameters of aqueous solution (salt concentration and pH). From the results, we found that 20-mer aptamer binding to thrombin by G-guadruplex structure and dominated by electrostatic interactions. The 29-mer aptamer binds to thrombin by hairpin structure and is driven by hydrophobic effects. Furthermore, we comfirmed this argument by Isothermal Titration Calometry measurements . By experimental results, we suggested that the structure of these two aptamers is an important factor to cause the different binding mechanism between these two aptamers with thrombin.
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Huang, Chi-Chieh, i 黃繼傑. "Real-Time Detection of α-Thrombin Binding to Single-Strand DNA Aptamers and Dengue Virus DNA Hybridization by ARROW-B SPR Biosensors". Thesis, 2008. http://ndltd.ncl.edu.tw/handle/73575633967046751075.
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碩士國立交通大學
電子工程系所
96
In this thesis, an antiresonant reflecting optical waveguide of type B (ARROW-B) surface plasmon resonance (SPR) biosensor operating in the aqueous environment has been investigated. The ARROW-B SPR biosensor is proposed to provide a label-free, high-throughput and highly surface-sensitive platform to detect the bimolecular interactions in real time. The design and fabrication process of the ARROW-B SPR sensor chips are described and discussed. Besides, the primary analytes for the bioassay experiments are divided into two categories based on different binding characteristics. First, the real-time detection of α-thrombin binding to ssDNA aptamers was under in-depth investigation. The gold nanoparticles modified with anti-thrombin antibodies were employed to bind to the α-thrombins for signal amplification. The detection limit of this biosensor to α-thrombin was measured at 1 pM level, which was comparable to that of the Biacore 3000 system but at much lower cost. Second, the real-time detection of dengue virus ssDNA hybridization was studied. The dengue virus DNA probe was modified with a thiol group at one end to achieve effective immobilization on the Au surface, while the DNA target utilized the complementary sequence to bind to the immobilized probe. In summary, the measurement results have shown that the ARROW-B SPR biosensors can be applied to detect the ssDNA aptamer/α-thrombin interaction and dengue virus ssDNA hybridization both quantitatively and qualitatively in real time.
Części książek na temat "Thrombin binding aptamer"
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Kumar, Jangam Vikram, Wen-Yih Chen, Jeffrey J. P. Tsai i Wen-Pin Hu. "Molecular Simulation Methods for Selecting Thrombin-Binding Aptamers". W Lecture Notes in Electrical Engineering, 743–49. Dordrecht: Springer Netherlands, 2013. http://dx.doi.org/10.1007/978-94-007-6996-0_78.
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Lamberti, Ilaria, Jan Rakitka, Tibor Hianik i Lucia Mosiello. "A Piezoelectric Quartz Crystal Sensor Applied for Thrombin-Binding Aptamers". W Lecture Notes in Electrical Engineering, 449–53. Dordrecht: Springer Netherlands, 2011. http://dx.doi.org/10.1007/978-94-007-1324-6_73.
Pełny tekst źródłaStreszczenia konferencji na temat "Thrombin binding aptamer"
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Ma, Xiao, i Pranav Shrotriya. "Study on Specific Binding Interaction Between Protein and DNA Aptamer via Dynamic Force Spectroscopy". W ASME 2013 2nd Global Congress on NanoEngineering for Medicine and Biology. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/nemb2013-93119.
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Recently the need to design nanoscale, sensitive and flexible bio-sensors or biotic-abiotic interface keeps increasing. One of the essential challenges on this objective is to grasp a thorough understanding of the mechanism governing binding interaction between bio-molecules. In this study we aim to demonstrate the binding specificity and reveal force interaction between the anti-coagulation protein thrombin and the single-stranded DNA thrombin aptamer by application of Atomic Force Microscopy (AFM). The thiolated aptamer was deposited onto gold substrate, and then repeatedly brought into contact with a thrombin-coated AFM tip, and force drop-offs during the pull-off were measured to determine the unbinding force between the thrombin-aptamer pair. The results from experiment show that the thrombin-aptamer pair has specific binding and the force between the pair exhibits loading rate dependence. It was shown that the binding forces of the thrombin-aptamer interaction increases with growth of loading rates. The average binding force for a single thrombin/aptamer pair increased from 20 pN to 40 pN, with loading rate changes from 500pN/s to 13500pN/s. Distribution of the unbinding forces measured for each loading rate can be explained on the basis of single energy barrier model for molecular bond breakage.
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Riccardi, Claudia, Albert Meyer, Jean-Jacques Vasseur, Irene Russo Krauss, Luigi Paduano, François Morvan i Daniela Montesarchio. "Cyclic and pseudocyclic thrombin binding aptamer analogues as improved anticoagulant agents". W 7th International Electronic Conference on Medicinal Chemistry. Basel, Switzerland: MDPI, 2021. http://dx.doi.org/10.3390/ecmc2021-11595.
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Chen, Kok Hao, i Jong Hyun Choi. "DNA Oligonucleotide-Templated Nanocrystals: Synthesis and Novel Label-Free Protein Detection". W ASME 2009 International Mechanical Engineering Congress and Exposition. ASMEDC, 2009. http://dx.doi.org/10.1115/imece2009-11958.
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Semiconductor and magnetic nanoparticles hold unique optical and magnetic properties, and great promise for bio-imaging and therapeutic applications. As part of their stable synthesis, the nanocrystal surfaces are usually capped by long chain organic moieties such as trioctylphosphine oxide. This capping serves two purposes: it saturates dangling bonds at the exposed crystalline lattice, and it prevents irreversible aggregation by stabilizing the colloid through entropic repulsion. These nanocrystals can be rendered water-soluble by either ligand exchange or overcoating, which hampers their widespread use in biological imaging and biomedical therapeutics. Here, we report a novel scheme of synthesizing fluorescent PbS and magnetic Fe3O4 nanoparticles using DNA oligonucleotides. Our method of PbS synthesis includes addition of Na2S to the mixture solution of DNA sequence and Pb acetate (at a fixed molar ratio of DNA/S2−/Pb2+ of 1:2:4) in a standard TAE buffer at room temperature in the open air. In the case of Fe3O4 particle synthesis, ferric and ferrous chloride were mixed with DNA in DI water at a molar ratio of DNA/Fe2+/Fe3+ = 1:4:8 and the particles were formed via reductive precipitation, induced by increasing pH to ∼11 with addition of ammonium hydroxide. These nanocrystals are highly stable and water-soluble immediately after the synthesis, due to DNA termination. We examined the surface chemistry between oligonucleotides and nanocrystals using FTIR spectroscopy, and found that the different chemical moieties of nucleobases passivate the particle surface. Strong coordination of primary amine and carbonyl groups provides the chemical and colloidal stabilities, leading to high particle yields (Figure 1). The resulting PbS nanocrystals have a distribution of 3–6 nm in diameter, while a broader size distribution is observed with Fe3O4 nanoparticles as shown in Figure 1b and c, respectively. A similar observation was reported with the pH change-induced Fe3O4 particles of a bimodal size distribution where superparamagnetic and ferrimagnetic magnetites co-exist. In spite of the differences, FTIR measurements suggest that the chemical nature of the oligonucleotide stabilization in this case is identical to the PbS system. As a particular application, we demonstrate that aptamer-capped PbS QD can detect a target protein based on selective charge transfer, since the oligonucleotide-templated synthesis can also serve the additional purpose of providing selective binding to a molecular target. Here, we use thrombin and a thrombin-binding aptamer as a model system. These QD have diameters of 3∼6 nm and fluoresce around 1050 nm. We find that a DNA aptamer can passivate near IR fluorescent PbS nanocrystals, rendering them water-soluble and stable against aggregation, and retain the secondary conformation needed to selectively bind to its target, thrombin, as shown in Figure 2. Importantly, we find that when the aptamer-functionalized nanoparticles binds to its target (only the target), there is a highly systematic and selective quenching of the PL, even in high concentrations of interfering proteins as shown in Figure 3a and b. Thrombin is detected within one minute with a detection limit of ∼1 nM. This PL quenching is attributed to charge transfer from functional groups on the protein to the nanocrystals. A charge transfer can suppress optical transition mechanisms as we observe a significant decrease in QD absorption with target addition (Figure 3c). Here, we rule out other possibilities including Forster resonance energy transfer (FRET) and particle aggregation, because thrombin absorb only in the UV, and we did not observe any significant change in the diffusion coefficient of the particles with the target analyte, respectively. The charge transfer-induced photobleaching of QD and carbon nanotubes was observed with amine groups, Ru-based complexes, and azobenzene compounds. This selective detection of an unlabeled protein is distinct from previously reported schemes utilizing electrochemistry, absorption, and FRET. In this scheme, the target detection by a unique, direct PL transduction is observed even in the presence of high background concentrations of interfering negatively or positively charged proteins. This mechanism is the first to selectively modulate the QD PL directly, enabling new types of label free assays and detection schemes. This direct optical transduction is possible due to oligonucleotidetemplated surface passivation and molecular recognition. This chemistry may lead to more nanoparticle-based optical and magnetic probes that can be activated in a highly chemoselective manner.
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Huang, Chi-Chieh, Hsin-Feng Hsu, Sz-Hau Chen, Kun-Yu Tsai, Yang-Tung Huang, Chih-Sheng Lin i Shih-Hsin Hsu. "Real-time detection of α-thrombin binding to single-strand DNA aptamers by a highly sensitive Si-based waveguide SPR biosensor". W Asia Pacific Optical Sensors Conference, redaktorzy John Canning i Gangding Peng. SPIE, 2012. http://dx.doi.org/10.1117/12.915947.
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