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Aptamer
Kinds of Aptamer Selected AbstractsAn Aptamer-Based Bound/Free Separation System for Protein DetectionELECTROANALYSIS, Issue 11 2009Mieko Fukasawa Abstract Aptamer hybridizes with its complementary strand. However, the complementary strand has difficulties to hybridize with the aptamer bound to a target because the aptamer forms higher-order structures. Exploiting this property, we developed simple bound/free separation systems for thrombin and IgE detection. The complementary strand was immobilized onto beads and the aptamer was labeled with pyrroquinoline quinone glucose dehydrogenase (PQQGDH). In the absence of a target, the aptamer is trapped by beads, whereas in the presence of a target, the aptamer bound to the target is not trapped. Thus the aptamer-target complexes can be recovered easily and detected by PQQGDH activity. This system allow the detection of 270,pM thrombin and 1,nM IgE. [source] Detection of C Reactive Protein (CRP) in Serum by an Electrochemical Aptamer-Based Sandwich AssayELECTROANALYSIS, Issue 11 2009Sonia Centi Abstract A disposable electrochemical assay involving magnetic particles and carbon-based screen-printed electrodes (SPCEs) was developed for the detection of C Reactive Protein (CRP). CRP is a plasma protein and is among the most expressed proteins in acute phase inflammation cases, being a known biomarker for inflammatory states. The assay was based on a sandwich format in which a RNA aptamer was coupled to a monoclonal antibody and alkaline phosphatase (AP) was used as enzymatic label. After the sandwich assay, the modified magnetic beads were captured by a magnet on the surface of a graphite working electrode and the electrochemical detection was thus achieved through the addition of the AP substrate (,-naphthyl-phosphate) and ,-naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry (DPV). The parameters influencing the different steps of the assay were optimized in order to reach the best sensitivity and specificity. With the optimized conditions, the assay was applied to the analysis of CRP free serum and serum samples. [source] Synthesis of Functionalized Au Nanoparticles for Protein Detection,ADVANCED MATERIALS, Issue 3 2008R. Jana Aptamer and antibody functionalized Au nanoparticles are synthesized and used for protein detection (see figure). These particles are highly water soluble and as small as 10 nm. However, they are enlarged after a protein binding event to enhance signal sensitivity. A conventional western blot protocol is used to enable detection of the proteins with nanomolar sensitivity, with the naked eye. [source] Aptamere als Therapeutika und als Werkzeuge zur Wirkstoffsuche.PHARMAZIE IN UNSERER ZEIT (PHARMUZ), Issue 6 2007Nukleinsäuren im Drug Discovery Prozess Aptamere sind potente Proteininhibitoren und leisten, wie kaum eine andere Substanzklasse, einen wichtigen Beitrag zum Drug-Discovery-Prozess. Mit Macugen® ist das erste Aptamer als Therapeutikum verfügbar und kann zur Behandlung der AMD eingesetzt werden. Neben ihrem direkten Einsatz als Therapeutika werden Aptamere sowohl zur Validierung pharmakologisch relevanter Proteine als auch zur Identifizierung niedermolekularer Substanzen verwandt. Die Aptamertechnologie repräsentiert eine Technologieplattform, deren vielseitige Einsatzgebiete von der funktionellen Charakterisierung von Biomolekülen bis hin zur Identifikation niedermolekularer Substanzen reichen. [source] Modulation der pri-miRNA-Reifung durch ein die apikale Schleife bindendes Aptamer,ANGEWANDTE CHEMIE, Issue 27 2010Christina Reife Leistung: RNA-Aptamere können als spezifische Moleküle die pri-miRNA-Reifung modulieren. Dieser Ansatz, mit dessen Hilfe die MikroRNA-Regulation gezielt beeinflusst werden kann, zeigt neue Wege bei der Entwicklung von Therapien auf. [source] Immediate Detection of Living Bacteria at Ultralow Concentrations Using a Carbon Nanotube Based Potentiometric Aptasensor,ANGEWANDTE CHEMIE, Issue 40 2009Gustavo So braucht's keinen Arzt: Ein Aptamer, das an eine mit einwandigen Kohlenstoffnanoröhren bedeckte Elektrode gebunden ist, wechselwirkt selektiv mit Bakterien (siehe Bild). Das daraus resultierende elektrochemische Signal ist sehr genau und reproduzierbar und setzt bei äußerst niedrigen Bakterienkonzentrationen ein. Damit handelt es sich um eine einfache, selektive Methode zum Nachweis von Pathogenen. [source] Reversible Cell-Specific Drug Delivery with Aptamer-Functionalized Liposomes,ANGEWANDTE CHEMIE, Issue 35 2009Zehui Cao Dr. Vorteil Aptamer: Eine zellspezifische Freisetzung des Tumortherapeutikums Cisplatin mithilfe eines Nucleolin-Aptamer-konjugierten, Cisplatin-verkapselnden Liposomsystems wird vorgestellt. Calcein wurde in die MCF-7-Zielzellen eingeschlossen (oberes Bild), nicht jedoch in LNCaP-Zellen (unteres Bild). Wesentlich ist zudem, dass das Ausmaß der Freisetzung mit einer zum Aptamer komplementären DNA als Antidot eingestellt werden kann. [source] Regulation of Thrombin Activity with a Bifunctional Aptamer and Hemin: Development of a New Anticoagulant and Antidote PairCHEMBIOCHEM, Issue 13 2009Jing Wang Through thick and thin: A bifunctional aptamer (BA) and hemin have been designed as an anticoagulant and antidote pair. BA, as anticoagulant, inhibits thrombin by forming a BA,thrombin complex. Because of the higher affinity between hemin and BA, once hemin is added, it replaces thrombin to form a BA,hemin complex. Thrombin is then released and reactivated to exhibit blood-clotting activity. [source] Controlling DNA Polymerization with a Switchable AptamerCHEMBIOCHEM, Issue 14 2007Eike Friedrichs Controllable biochemical reactions. DNA polymerization by Taq polymerase can be controlled by switching an aptamer for Taq Pol between a binding and a nonbinding form. [source] Multianalyte Electrochemical Biosensor Based on Aptamer- and Nanoparticle-Integrated Bio-Barcode AmplificationCHEMISTRY - AN ASIAN JOURNAL, Issue 2 2010Xuemei Li Dr. Abstract In the present work, a signal-on electrochemical sensing strategy for the simultaneous detection of adenosine and thrombin is developed based on switching structures of aptamers. An Au electrode as the sensing surface is modified with two kinds of thiolated capture probes complementary to the linker DNA that contains either an adenosine aptamer or thrombin aptamer. The capture probes hybridize with their corresponding linker DNA, which has prehybridized with the reporter DNA loaded onto the gold nanoparticles (AuNPs). The AuNP contained two kinds of bio-barcode DNA: one is complementary to the linker DNA (reporter), whereas the other is not (signal) and is tagged with different metal sulfide nanoparticles. Thus a "sandwich-type" sensing interface is fabricated for adenosine and thrombin. With the introduction of adenosine and thrombin, the aptamer parts bind with their targets and fold to form the complex structures. As a result, the bio-barcoded AuNPs are released into solution. The metal sulfide nanoparticles are measured by anodic stripping voltammetry (ASV), and the concentrations of adenosine and thrombin are proportional to the signal of either metal ion. With the dual amplification of the bio-barcoded AuNP and the preconcentration of metal ions through ASV technology, detection limits as low as 6.6×10,12,M for adenosine and 1.0×10,12,M for thrombin are achieved. The sensor exhibits excellent selectivity and detectability in biological samples. [source] An Aptamer-Based Bound/Free Separation System for Protein DetectionELECTROANALYSIS, Issue 11 2009Mieko Fukasawa Abstract Aptamer hybridizes with its complementary strand. However, the complementary strand has difficulties to hybridize with the aptamer bound to a target because the aptamer forms higher-order structures. Exploiting this property, we developed simple bound/free separation systems for thrombin and IgE detection. The complementary strand was immobilized onto beads and the aptamer was labeled with pyrroquinoline quinone glucose dehydrogenase (PQQGDH). In the absence of a target, the aptamer is trapped by beads, whereas in the presence of a target, the aptamer bound to the target is not trapped. Thus the aptamer-target complexes can be recovered easily and detected by PQQGDH activity. This system allow the detection of 270,pM thrombin and 1,nM IgE. [source] DNA Aptamers that Bind to PQQGDH as an Electrochemical Labeling ToolELECTROANALYSIS, Issue 11 2009Yuko Osawa Abstract We screened DNA aptamers that bind to pyrroquinoline quinone glucose dehydrogenase (PQQGDH) for the development of an electrochemical labeling tool. PQQGDH is an excellent enzyme for the signal amplification of biosensors. We focused on DNA aptamers as labeling agents and tried to select those DNA aptamers that bind to PQQGDH without affecting its enzymatic activity. After 7 rounds of screening, one aptamer was obtained: ,PGa4'. It bound to PQQGDH with specificity and showed no effect on the glucose dehydrogenase (GDH) activity. Moreover, beads labeled with PQQGDH via PGa4 generated an electrical current upon glucose addition. Therefore, we believe that the PGa4 aptamer against PQQGDH may become a powerful labeling tool for electrochemical biosensors. [source] Detection of C Reactive Protein (CRP) in Serum by an Electrochemical Aptamer-Based Sandwich AssayELECTROANALYSIS, Issue 11 2009Sonia Centi Abstract A disposable electrochemical assay involving magnetic particles and carbon-based screen-printed electrodes (SPCEs) was developed for the detection of C Reactive Protein (CRP). CRP is a plasma protein and is among the most expressed proteins in acute phase inflammation cases, being a known biomarker for inflammatory states. The assay was based on a sandwich format in which a RNA aptamer was coupled to a monoclonal antibody and alkaline phosphatase (AP) was used as enzymatic label. After the sandwich assay, the modified magnetic beads were captured by a magnet on the surface of a graphite working electrode and the electrochemical detection was thus achieved through the addition of the AP substrate (,-naphthyl-phosphate) and ,-naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry (DPV). The parameters influencing the different steps of the assay were optimized in order to reach the best sensitivity and specificity. With the optimized conditions, the assay was applied to the analysis of CRP free serum and serum samples. [source] Electrochemical Aptasensor for the Determination of Cocaine Incorporating Gold Nanoparticles ModificationELECTROANALYSIS, Issue 13 2008Xiaoxia Li Abstract A novel electrochemical aptasensor incorporating a signal enhancement for the determination of cocaine was designed. Gold nanoparticles were self-assembled onto the surface of a gold electrode through 1,6-hexanedithiol. A bifunctional derivative of the 32-base cocaine-binding aptamer with a redox-active ferrocene moiety and a thiol linker group at the termini of the strand was self-assembled onto the surface of gold nanoparticles. The oxidation peak current is linearly related to the concentration of cocaine from 1.0 to 15.0,,M with a detection limit of 0.5,,M. It was found that the sensitivity of the aptasensor with gold nanoparticles modification was ca. 10-fold higher than that of the aptasensor without gold nanoparticles modification. This work demonstrates that gold nanoparticles-assembled gold electrode provides a promising platform for immobilizing aptamer and enhancing the sensitivity. [source] High-sensitivity detection of oxytetracycline using light scattering agglutination assay with aptasensorELECTROPHORESIS, Issue 18 2010Keesung Kim Abstract We present an aptamer-based biosensor (aptasensor) for rapid and high-sensitive detection of oxytetracycline (OTC) antibiotic in PBS inside a Y-channel PDMS microfluidic device. The detection was made by real-time monitoring of the agglutination assay of ssDNA aptamer-conjugated polystyrene latex microspheres with proximity optical fibers. The agglutination assay was performed with serially diluted OTC antibiotic solutions using highly carboxylated polystyrene particles of 920,nm diameter conjugated with OTC-binding ssDNA aptamer. Proximity optical fibers were used to measure the increase in 45° forward light scattering of the aggregated particles by fixing them around the viewing cell of the device with stable angle and distance to the detector. The detection limit was around 100,ppb for the current aptasensor system with the detection time less than 3,min. [source] Interaction study of a lysozyme-binding aptamer with mono- and divalent cations by ACEELECTROPHORESIS, Issue 3 2010Marie Girardot Abstract Binding between an aptamer and its target is highly dependent on the conformation of the aptamer molecule, this latter seeming to be affected by a variety of cations. As only a few studies have reported on the interactions of monovalent or divalent cations with aptamers, we describe herein the use of ACE in its mobility shift format for investigating interactions between various monovalent (Na+, K+, Cs+) or divalent (Mg2+, Ca2+, Ba2+) cations and a 30-mer lysozyme-binding aptamer. This study was performed in BGEs of different natures (phosphate and MOPS buffers) and ionic strengths. First, the effective charges of the aptamer in 30,mM ionic strength phosphate and MOPS (pH 7.0) were estimated to be 7.4 and 3.6, respectively. Then, corrections for ionic strength and counterion condensation effects were performed for all studies. The effective mobility shift was attributed not only to these effects, but also to a possible interaction with the buffer components (binary or ternary complexes) as well as possible conformational changes of the aptamer. Finally, apparent binding constants were calculated for divalent cations with mathematical linearization methods, and the influence of the nature of the BGE was evidenced. [source] High-sensitive determination of human ,-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresisELECTROPHORESIS, Issue 12 2008Yilin Li Abstract ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5,nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400,nM and the LOD was 2,nM (74,ng/mL, S/N,=,3), i.e., 40,amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin. [source] Microaffinity purification of proteins based on photolytic elution: Toward an efficient microbead affinity chromatography on a chipELECTROPHORESIS, Issue 3 2005Woo-Jae Chung Abstract A bead affinity chromatography system, which was based on the photolytic elution method, was integrated into a glass-silicon microchip to purify specific target proteins. CutiCore® beads, which were coupled with a photo-cleavable ligand, such as biotin and an RNA aptamer, were introduced into a filter chamber in the microchip. The protein mixture containing target protein labeled with fluorescein isothiocyanate (FITC) was then passed through the packed affinity beads in the microchamber by pressure-driven flow. During the process, the adsorbed protein on the bead was monitored by fluorescence. The concentrated target protein on the affinity bead was released by simple irradiation with UV light at a wavelength of 360 nm, and subsequently eluted with the phosphate buffer flow. The eluted target protein was quantitatively detected via the fluorescence intensity measurements at the downstream of the capillary connected to the outlet of the microchip. The microaffinity purification allowed for a successful method for the identification of specific target proteins from a protein mixture. In addition, the feasibility of this system for use as a diagnosis chip was demonstrated. [source] Protein Detecting Arrays Based on Cationic Polythiophene,DNA-Aptamer Complexes,ADVANCED MATERIALS, Issue 20 2006Abérem, M. Béra By combining an appropriate DNA aptamer with a cationic polythiophene optical transducer, human thrombin can be specifically detected on microarrays in the attomole range in less than one hour without any tagging of the target. The system can be modified and utilized as a probe for the detection of various proteins or other biomolecules. This work opens new interesting possibilities for simple and rapid multiparametric analysis in genomics and proteomics. [source] Label free optical sensor for Avidin based on single gold nanoparticles functionalized with aptamersJOURNAL OF BIOPHOTONICS, Issue 4 2009Frank Jeyson Hernandez Abstract Optical spectroscopy of a single gold nanoparticle, functionalized with an aptamer, is used to sense the specific binding of avidin. Herewith, the field of single noble metal nanoparticle biosensors is extended to the important field of aptamer based assays. The sensitivity of this initial, but not yet optimized apta-nano-sensor is in the range of 20 nM. Due to its nanoscopic size, this single nanoparticle based apta-sensor may be used in nanoscopic volumes such as in array type assays or even inside cells. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] DNA aptamers developed against a soman derivative cross-react with the methylphosphonic acid core but not with flanking hydrophobic groupsJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2009John G. Bruno Abstract Twelve rounds of systematic evolution of ligands by exponential enrichment (SELEX) were conducted against a magnetic bead conjugate of the para -aminophenylpinacolylmethylphosphonate (PAPMP) derivative of the organophosphorus (OP) nerve agent soman (GD). The goal was to develop DNA aptamers that could scavenge GD in vivo, thereby reducing or eliminating the toxic effects of this dangerous compound. Aptamers were sequenced and screened in peroxidase-based colorimetric plate assays after rounds 8 and 12 of SELEX. The aptamer candidate sequences exhibiting the highest affinity for the GD derivative from round 8 also reappeared in several clones from round 12. Each of the highest affinity PAPMP-binding aptamers also bound methylphosphonic acid (MPA). In addition, the aptamer with the highest overall affinity for PAPMP carried a sequence motif (TTTAGT) thought to bind MPA based on previously published data (J. Fluoresc 18: 867,876, 2008). This sequence motif was found in several other relatively high affinity PAPMP aptamer candidates as well. In studies with the nerve agent GD, pre-incubation of a large molar excess of aptamer candidates failed to protect human butyrylcholinesterase (BuChE) from inhibition. With the aid of three-dimensional molecular modeling of the GD derivative it appears that a hydrophilic cleft sandwiched between the pinacolyl group and the p -aminophenyl ring might channel nucleotide interactions to the phosphonate portion of the immobilized GD derivative. However, bona fide GD free in solution may be repulsed by the negative phosphate backbone of aptamers and rotate its phosphonate and fluorine moieties away from the aptamer to avoid being bound. Future attempts to develop aptamers to GD might benefit from immobilizing the pinacolyl group of bona fide GD to enhance exposure of the phosphonate and fluorine to the random DNA library. Copyright © 2008 John Wiley & Sons, Ltd. [source] Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factor and malachite greenJOURNAL OF MOLECULAR RECOGNITION, Issue 2 2009Xing Zhang Abstract In previous work, Vibrio proteolyticus 5S rRNA was shown to stabilize 13,50 nucleotide "guest" RNA sequences for expression in Escherichia coli. The expressed chimeric RNAs accumulated to high levels in E. coli without being incorporated into ribosomes and without obvious effects on the host cells. In this work, we inserted sequences encoding known aptamers recognizing a protein and an organic dye into the 5S rRNA carrier and showed that aptamer function is preserved in the chimeras. A surface plasmon resonance competitive binding assay demonstrated that a vascular endothelial growth factor (VEGF) aptamer/5S rRNA chimera produced in vitro by transcriptional runoff could compete with a DNA aptamer for VEGF, implying binding of the growth factor by the VEGF "ribosomal RNA aptamer." Separately, a 5S rRNA chimera displaying an aptamer known to increase the fluorescence of malachite green (MG) also enhanced MG fluorescence. Closely related control rRNA molecules showed neither activity. The MG aptamer/5S rRNA chimera, like the original MG aptamer, also increased the fluorescence of other triphenyl methane (TPM) dyes such as crystal violet, methyl violet, and brilliant green, although less effectively than with MG. These results indicate that the molecular recognition properties of aptamers are not lost when they are expressed in the context of a stable 5S rRNA carrier. Inclusion of the aptamer in a carrier may facilitate production of large quantities of RNA aptamers, and may open an approach to screening aptamer libraries in vivo. Copyright © 2009 John Wiley & Sons, Ltd. [source] Raman and surface-enhanced Raman spectroscopic studies of the 15-mer DNA thrombin-binding aptamerJOURNAL OF RAMAN SPECTROSCOPY, Issue 3 2010Cynthia V. Pagba Abstract Aptamers are single-stranded oligonucleotides that selectively bind to their target molecules owing to their ability to form secondary structures and shapes. The 15-mer (5,-GGTTGGTGTGGTTGG-3,) DNA thrombin-binding aptamer (TBA) binds to thrombin following the formation of a quadruplex structure via the Hoogsten-type G,G interactions. In the present study, Raman and SERS spectra of TBA and thiolated TBA (used to facilitate covalent bonding to metal nanoparticle) in different conditions are investigated. The spectra of the two analogs exhibit vibrations, such as the C8N7H2 deformation band at ,1480 cm,1 of the guanine tetrad, that are characteristic of the quadruplex structure in the presence of K+ ions or at low temperature. Interestingly, SERS spectra of the two analogs differ markedly from their respective normal Raman spectra, possibly due to changes in the conformation of the aptamer upon binding, as well as to the specific interaction of individual vibrational modes with the metal surface. In addition, the SERS spectra of the thiolated aptamer show significant changes with different concentrations, which may be due to different orientation of the molecule with respect to the metal surface. This study provides useful information for the development of label-free aptamer-based SERS sensors and assays. Copyright © 2009 John Wiley & Sons, Ltd. [source] Competitive affinity capillary electrophoresis assay based on a "hybrid" pre-incubation/on-capillary mixing format using an enantioselective aptamer as affinity ligandJOURNAL OF SEPARATION SCIENCE, JSS, Issue 12 2008Josephine Ruta Abstract In this paper, we describe an aptamer-based competitive affinity CE (ACE) assay involving (i) the pre-incubation of the target (D-arginine) and the specific ligand (anti-D-arginine-L-RNA aptamer) before (ii) the competition with the labeled target (dansylated D-arginine) through an on-capillary mixing strategy. The effects of some critical operating parameters such as the applied voltage and the sample-aptamer mixture plug length on the assay sensitivity were investigated. The ACE assay appeared particularly dependent on the plug length of the pre-incubated sample-aptamer solution. It was shown that this "hybrid" strategy significantly improved the assay sensitivity relative to that obtained with a "full" on-capillary mixing approach. [source] 4-Thio-deoxyuridylate-modified thrombin aptamer and its inhibitory effect on fibrin clot formation, platelet aggregation and thrombus growth on subendothelial matrixJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2008S. MENDELBOUM RAVIV Summary.,Background:,The consensus thrombin aptamer C15-mer is a single-stranded DNA of 15 nucleotides [d(GGTTGGTGTGGTTGG)] that was identified by the selection of thrombin-binding molecules from a large combinatorial library of oligonucleotides. It is capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite 1. As has been shown in our earlier studies, the 4-thio-deoxyuridylate (s4dU)-containing oligonucleotides have high affinity for a number of proteins, due to the reduced hydrophilic character of the modified oligonucleotide. Methods:,Three different analogs of the original thrombin-inhibiting sequence, in which some of the thymidylate residues were replaced by 4-thio-deoxyuridylates, were synthesized. The inhibitory effect of modified aptamers was tested on thrombin-catalyzed fibrin clot formation and fibrinopeptide A release from fibrinogen, thrombin-induced platelet aggregation/secretion, and the formation of thrombus on coverslips coated with human collagen type III, thrombin-treated fibrinogen or subendothelial matrix of human microvascular endothelial cells. Results:,As compared with the C15-mer, the analog with the sequence GG(s4dU)TGG(s4dU)G(s4dU)GGT(s4dU)GG (UC15-mer) showed a 2-fold increased inhibition of thrombin-catalyzed fibrin clot formation, fibrinopeptide A release, platelet aggregation and secretion in human plasma and thrombus formation on thrombin-treated fibrinogen surfaces under flow conditions. Concerning the inhibition of thrombin-induced fibrin formation from purified fibrinogen and activation of washed platelets, UC15-mer was 3-fold and twelve-fold more effective than C15-mer, respectively. Conclusion:,The replacement of four thymidylate residues in C15-mer by 4-thio-deoxyuridylate resulted in a new thrombin aptamer with increased anticoagulant and antithrombotic properties. [source] Crystallization and preliminary X-ray analysis of the complex of human ,-thrombin with a modified thrombin-binding aptamerACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Irene Russo Krauss The thrombin-binding aptamer (TBA) is a consensus DNA 15-mer that binds specifically to human ,-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. [source] Expression, crystallization and preliminary crystallographic analysis of RNA-binding protein Hfq (YmaH) from Bacillus subtilis in complex with an RNA aptamerACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Seiki Baba The Hfq protein is a hexameric RNA-binding protein which regulates gene expression by binding to RNA under the influence of diverse environmental stresses. Its ring structure binds various types of RNA, including mRNA and sRNA. RNA-bound structures of Hfq from Escherichia coli and Staphylococcus aureus have been revealed to have poly(A) RNA at the distal site and U-rich RNA at the proximal site, respectively. Here, crystals of a complex of the Bacillus subtilis Hfq protein with an A/G-repeat 7-mer RNA (Hfq,RNA) that were prepared using the hanging-drop vapour-diffusion technique are reported. The type 1 Hfq,RNA crystals belonged to space group I422, with unit-cell parameters a = b = 123.70, c = 119.13,Å, while the type 2 Hfq,RNA crystals belonged to space group F222, with unit-cell parameters a = 91.92, b = 92.50, c = 114.92,Å. Diffraction data were collected to a resolution of 2.20,Å from both crystal forms. The hexameric structure of the Hfq protein was clearly shown by self-rotation analysis. [source] Crystallization and preliminary X-ray diffraction data of an LNA 7-mer duplex derived from a ricin aptamerACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Charlotte Förster Locked nucleic acids (LNAs) are modified nucleic acids which contain a modified sugar such as , - d -2,- O,4,- C methylene-bridged ribofuranose or other sugar derivatives in LNA analogues. The ,- d -2,- O,4,- C methylene ribofuranose LNAs in particular possess high stability and melting temperatures, which makes them of interest for stabilizing the structure of different nucleic acids. Aptamers, which are DNAs or RNAs targeted against specific ligands, are candidates for substitution with LNAs in order to increase their stability. A 7-mer helix derived from the terminal part of an aptamer that was targeted against ricin was chosen. The ricin aptamer originally consisted of natural RNA building blocks and showed high affinity in ricin binding. For future stabilization of the aptamer, the terminal helix has been constructed as an `all-locked' LNA and was successfully crystallized in order to investigate its structural properties. Optimization of crystal growth succeeded by the use of different metal salts as additives, such as CuCl2, MgCl2, MnCl2, CaCl2, CoCl2 and ZnSO4. Preliminary X-ray diffraction data were collected and processed to 2.8,Å resolution. The LNA crystallized in space group P65, with unit-cell parameters a = 50.11, b = 50.11, c = 40.72,Å. The crystals contained one LNA helix per asymmetric unit with a Matthews coefficient of 3.17,Å3,Da,1, which implies a solvent content of 70.15%. [source] Mechanism-Guided Library Design and Dual Genetic Selection of Synthetic OFF RiboswitchesCHEMBIOCHEM, Issue 14 2009Norihito Muranaka Dr. Abstract After the recent discovery of bacterial riboswitches, synthetic riboswitches have been engineered by using natural and artificial RNA aptamers. In contrast to natural riboswitches, the majority of synthetic riboswitches in bacteria reported to date are ON switches that activate gene expression in response to the aptamer ligand. In this study, we adopted a mechanism-guided approach to design libraries predisposed to contain OFF riboswitches that respond to thiamine pyrophosphate (TPP). The first library design exploited a pseudo-Shine-Dalgarno (SD) sequence located near the 3,-end of the TPP aptamer, which would be less accessible to the ribosome when the aptamer is bound to TPP. In the second library, an SD sequence was strategically placed in the aptamer's P1 stem, which is stabilized upon ligand binding. OFF riboswitches were obtained by dual genetic selection of these libraries. The results underscore the importance of effective library design to achieve desired riboswitch functions. [source] Regulation of Thrombin Activity with a Bifunctional Aptamer and Hemin: Development of a New Anticoagulant and Antidote PairCHEMBIOCHEM, Issue 13 2009Jing Wang Through thick and thin: A bifunctional aptamer (BA) and hemin have been designed as an anticoagulant and antidote pair. BA, as anticoagulant, inhibits thrombin by forming a BA,thrombin complex. Because of the higher affinity between hemin and BA, once hemin is added, it replaces thrombin to form a BA,hemin complex. Thrombin is then released and reactivated to exhibit blood-clotting activity. [source] | ||||||||||||||||||||||||||||