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ABSTRACT: Lymphatic filariasis and onchocerciasis are severe diseases caused by filarial worms and affect more than 150 million people worldwide. Endosymbiotic α-proteobacteria Wolbachia are essential for these parasites throughout their life cycle. Using a high-throughput chemical screen, we identified a benzimidazole compound, wALADin1, that selectively targets the δ-aminolevulinic acid dehydratase (ALAD) of Wolbachia (wALAD) and exhibits macrofilaricidal effects on Wolbachia-containing filarial worms in vitro. wALADin1 is a mixed competitive/noncompetitive inhibitor that interferes with the Mg(2+)-induced activation of wALAD. This mechanism inherently excludes activity against the Zn(2+)-dependent human ortholog and might be translatable to Mg(2+)-responsive orthologs of other bacterial or protozoan pathogens. The specificity profile of wALADin1 derivatives reveals chemical features responsible for inhibitory potency and species selectivity. Our findings validate wALADins as a basis for developing potent leads that meet current requirements for antifilarial drugs.
Chemistry & biology 02/2013; 20(2):177-87. · 6.52 Impact Factor
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Eileen Magbanua,
Tijana Zivkovic,
Björn Hansen,
Niklas Beschorner,
Cindy Meyer,
Inken Lorenzen,
Joachim Grötzinger,
Joachim Hauber,
Andrew E Torda, Günter Mayer,
Stefan Rose-John,
Ulrich Hahn
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ABSTRACT: Aptamers are oligonucleotides that bind targets with high specificity and affinity. They have become important tools for biosensing, target detection, drug delivery and therapy. We selected the quadruplex-forming 16-mer DNA aptamer AID-1 [d(GGGT) 4] with affinity for the interleukin-6 receptor (IL-6R) and identified single nucleotide variants that showed no significant loss of binding ability. The RNA counterpart of AID-1 [r(GGGU) 4] also bound IL-6R as quadruplex structure. AID-1 is identical to the well-known HIV inhibitor T30923, which inhibits both HIV infection and HIV-1 integrase. We also demonstrated that IL-6R specific RNA aptamers not only bind HIV-1 integrase and inhibit its 3' processing activity in vitro, but also are capable of preventing HIV de novo infection with the same efficacy as the established inhibitor T30175. All these aptamer target interactions are highly dependent on formation of quadruplex structure.
RNA biology 12/2012; 10(2). · 5.56 Impact Factor
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ABSTRACT: A most able label: Labeled aptamers can be cross-linked to their target structures in a light-dependent and highly specific manner as a result of a new strategy termed aptamer-based affinity labeling (ABAL) of proteins. The aptamer-protein complexes can be enriched in vitro, from a cellular lysate and from the surface of living cells, opening new ways to study aptamer interactions in biological contexts.
Angewandte Chemie International Edition 08/2012; 51(36):9176-80. · 13.45 Impact Factor
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ABSTRACT: Spatial and temporal control over chemical and biological processes plays a key role in life, where the whole is often much more than the sum of its parts. Quite trivially, the molecules of a cell do not form a living system if they are only arranged in a random fashion. If we want to understand these relationships and especially the problems arising from malfunction, tools are necessary that allow us to design sophisticated experiments that address these questions. Highly valuable in this respect are external triggers that enable us to precisely determine where, when, and to what extent a process is started or stopped. Light is an ideal external trigger: It is highly selective and if applied correctly also harmless. It can be generated and manipulated with well-established techniques, and many ways exist to apply light to living systems--from cells to higher organisms. This Review will focus on developments over the last six years and includes discussions on the underlying technologies as well as their applications.
Angewandte Chemie International Edition 07/2012; 51(34):8446-76. · 13.45 Impact Factor
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ABSTRACT: 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 αF-αG 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.
Structure 06/2012; 20(8):1300-9. · 6.35 Impact Factor
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ABSTRACT: We have synthesized a light-activatable ("caged") derivative of glucosamine-6-phosphate (GlcN6P), which only upon irradiation becomes a cofactor for the glmS riboswitch. This glmS riboswitch maintains its activity when embedded in the 3'-untranslated region of eukaryotic mRNA molecules and caged GlcN6P reduces the amount of translated EGFP upon irradiation with light in vitro.
Photochemical and Photobiological Sciences 03/2012; 11(3):489-92. · 2.58 Impact Factor
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ABSTRACT: 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 progenitor molecule was obtained through domain reassembly.
ChemBioChem 02/2012; 13(5):631-4. · 3.94 Impact Factor
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ABSTRACT: Aptamers represent an emerging strategy to deliver cargo molecules, including dyes, drugs, proteins or even genes, into specific target cells. Upon binding to specific cell surface receptors aptamers can be internalized, for example by macropinocytosis or receptor mediated endocytosis. Here we report the in vitro selection and characterization of RNA aptamers with high affinity (Kd = 20 nM) and specificity for the human IL-6 receptor (IL-6R). Importantly, these aptamers trigger uptake without compromising the interaction of IL-6R with its natural ligands the cytokine IL-6 and glycoprotein 130 (gp130). We further optimized the aptamers to obtain a shortened, only 19-nt RNA oligonucleotide retaining all necessary characteristics for high affinity and selective recognition of IL-6R on cell surfaces. Upon incubation with IL-6R presenting cells this aptamer was rapidly internalized. Importantly, we could use our aptamer, to deliver bulky cargos, exemplified by fluorescently labeled streptavidin, into IL-6R presenting cells, thereby setting the stage for an aptamer-mediated escort of drug molecules to diseased cell populations or tissues.
RNA biology 01/2012; 9(1):67-80. · 5.56 Impact Factor
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ABSTRACT: We report on the direct electrochemical detection of aptamer-protein interactions, namely between a DNA aptamer and lysozyme (LYS) based on electrochemical impedance spectroscopy (EIS) technique. First, the affinity of the aptamer to LYS and control proteins was presented by using filter retention assay. An amino-modified version of the DNA aptamer-recognizing lysozyme was covalently immobilized on the surface of multiwalled carbon nanotube-modified screen-printed electrodes (MWCNT-SPEs), which were employed for measurements and have improved properties compared with bare SPEs. This carbon nanotube setup enabled the reliable monitoring of the interaction of lysozyme with its cognate aptamer by EIS transduction of the resistance to charge transfer (R(ct)) in the presence of 2.5 mM [Fe(CN)₆]³⁻/⁴⁻. This assay system provides a means for the label-free, concentration-dependent, and selective detection of lysozyme with an observed detection limit of 12.09 μg/ml (equal to 862 nM).
Analytical Biochemistry 12/2011; 421(2):454-9. · 3.00 Impact Factor
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ABSTRACT: Retooling RNA: RNA aptamers are high-affinity ligands that can be assembled with other structures to yield multivalent molecules. These properties have been addressed in two recent studies: One describes a GFP-like RNA reporter used to study the dynamics of endogenous RNA; the other study reports on an aptamer-templated assembly of multi-enzyme complexes in bacteria for the controlled production of secondary molecules (see picture).
Angewandte Chemie International Edition 11/2011; 50(52):12400-1. · 13.45 Impact Factor
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ABSTRACT: While many diagnostic assay platforms enable the measurement of analytes with high sensitivity, most of them result in a disruption of the analyte's native structure and, thus, in loss of function. Consequently, the analyte can be used neither for further analytical assessment nor functional analysis. Herein we report the use of caged aptamers as templates during apta-PCR analysis of targets. Aptamers are short nucleic acids that fold into a well-defined three-dimensional structure in which they interact with target molecules with high affinity and specificity. Nucleic acid aptamers can also serve as templates for qPCR approaches and, thus, have been used as high affinity ligands to bind to target molecules and subsequently for quantification by qPCR, an assay format coined apta-PCR. Caged aptamers in turn refer to variants that bear one or more photolabile groups at strategic positions. The activity of caged aptamers can thus be turned on or off by light irradiation. The latter allows the mild elution of target-bound aptamers while the target's native structure and function remain intact. We demonstrate that this approach allows the quantitative and subsequently the functional assessment of analytes. Since caged aptamers can be generated emanating from virtually every available aptamer, the described approach can be generalized and adopted to any target-aptamer pair and, thus, have a broad applicability in proteomics and clinical diagnostics.
ACS Chemical Biology 11/2011; 7(2):360-6. · 6.45 Impact Factor
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ABSTRACT: Aptamers comprise a range of molecular recognition scaffolds that can be engineered to bind to a legion of different proteins and other targets with excellent specificity and affinity. Because these non-natural oligonucleotides are accessible entirely synthetically, aptamers can be equipped with all sorts of reporter groups and can be coupled to many different carriers, surfaces, nanoparticles, or other biomolecules. They can be used in a highly modular fashion and often recognize their targets by a mechanism in which the aptamer undergoes considerable structural rearrangement, which can be exploited for transducing a binding event into a signal. As a consequence, aptamers have been adapted to a huge variety of "read-out configurations" and are increasingly used as capture agents in many different bioanalytical methods. But despite considerable success with these applications, many remaining challenges must still be overcome for the more widespread incorporation of aptasensors in clinical and environmental biosensing and diagnostics to take place. Some particularly noteworthy progress on this front is currently being made with aptasensor configurations that can be used for the multiplexed sensing of many analytes in parallel. In this Account, we describe some of the concepts involved in transducing the binding of a ligand into a signal through various physico-chemical interactions. Research in this area usually involves the combination of the molecular biology of proteins and nucleic acids with biotechnology, synthetic chemistry, physical chemistry, and surface physics. We begin with a brief introduction of the properties and characteristics that qualify aptamers as capture agents for many different analytes and their suitability as highly versatile biosensor components. We then address approaches that apply to surface acoustic wave configurations, drawing largely from our own contributions to aptasensor development, before moving on to describe previous and recent progress in multiplexed aptasensors. Obtaining proteome-wide profiles in cells, organs, organisms, or full populations requires the ability to accurately measure many different analytes in small sample volumes over a broad dynamic range. Multiplexed sensing is an invaluable tool in this endeavor. We discuss what we consider the biggest obstacles to the broader clinical use of aptasensor-based diagnostics and our perspective on how they can be surmounted. Finally,we explore the tremendous potential of aptamer-based sensors that can specifically discriminate between diseased and healthy cells. Progress in these areas will greatly expand the range of aptasensor applications, leading to enhanced diagnosis of diseases in clinical practice and, ultimately, improved patient care.
Accounts of Chemical Research 08/2011; 44(12):1349-58. · 21.64 Impact Factor
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Angewandte Chemie International Edition 06/2011; 50(27):6075-8. · 13.45 Impact Factor
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ABSTRACT: The glmS-riboswitch is unique among riboswitch families as it represents a metabolite-dependent ribozyme that undergoes self-cleavage upon recognition of glucosamin-6-phosphate. The glmS-riboswitch is located in the 5'-untranslated region of bacterial genes involved in cell wall biosynthesis. Therefore, this riboswitch represents a promising target for developing new antibiotics. We describe the metabolite-dependent glmS-riboswitch of pathologically relevant and vancomycin-resistant Staphylococcus aureus and the discovery and synthesis of a carba-sugar with potency similar to that of the native metabolite glucosamine-6-phosphate in modulating riboswitch activity. This compound represents a valuable lead structure for the development of antibiotics with a novel mode of action.
ACS Chemical Biology 04/2011; 6(7):675-8. · 6.45 Impact Factor
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ABSTRACT: Ribonucleic acid (RNA) is a multifunctional type of molecule, playing critical roles in protein biosynthesis and regulation. In recent years, suppression of protein translation by so-called microRNAs came into the focus of research, especially because deregulation of this process has been shown to play a role in malignant transformation. Furthermore, RNA molecules circulating in the blood have been revealed as a novel class of markers for diagnosis of cancers. Moreover, genetic information of some pathogens is stored as RNA, allowing their sensitive detection using nucleic acid amplification techniques. In this article, the principle of detecting different RNA types by real-time reverse-transcription polymerase chain reaction applications is described. Furthermore, the emerging use of microRNA and circulating RNA profiles complementing the broad spectrum of RNA diagnosis is discussed.
WIREs RNA 01/2011; 2(1):32-41.
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Marc Beyer,
Yasser Thabet,
Roman-Ulrich Müller,
Timothy Sadlon,
Sabine Classen,
Katharina Lahl,
Samik Basu,
Xuyu Zhou,
Samantha L Bailey-Bucktrout,
Wolfgang Krebs, [......],
Michael Famulok,
Percy A Knolle,
Claudia Wickenhauser,
Waldemar Kolanus,
Bernhard Schermer,
Jeffrey A Bluestone,
Simon C Barry,
Tim Sparwasser,
James L Riley,
Joachim L Schultze
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ABSTRACT: Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.
Nature Immunology 01/2011; 12(9):898-907. · 26.01 Impact Factor
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ABSTRACT: Aptamers that target a specific cell subpopulation within composite mixtures represent invaluable tools in biomedical research and in the development of cell-specific therapeutics. Here we describe a detailed protocol for a modular and generally applicable scheme to select aptamers that target the subpopulations of cells in which you are interested. A fluorescence-activated cell-sorting device is used to simultaneously differentiate and separate those subpopulations of cells having bound and unbound aptamers. There are fewer false positives when using this approach in comparison with other cell-selection approaches in which unspecific binding of nucleic acids to cells with reduced membrane integrity or their unselective uptake by dead cells occurs more often. The protocol provides a state-of-the-art approach for identifying aptamers that selectively target virtually any cell type under investigation. As an example, we provide the step-by-step protocol targeting CD19(+) Burkitt's lymphoma cells, starting from the pre-SELEX (systematic evolution of ligands by exponential amplification) measurements to establish suitable SELEX conditions and ending at completion of the SELEX procedure, which reveals the enriched single-stranded DNA library.
Nature Protocol 12/2010; 5(12):1993-2004. · 8.36 Impact Factor
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ABSTRACT: Functional nucleic acids, such as aptamers and allosteric ribozymes, can sense their ligands specifically, thereby undergoing structural alterations that can be converted into a detectable signal. The direct coupling of molecular recognition to signal generation enables the production of versatile reporters that can be applied as molecular probes for various purposes, including high-throughput screening. Here we describe an unprecedented type of a nucleic acid-based sensor system and show that it is amenable to high-throughput screening (HTS) applications. The approach detects the displacement of an aptamer from its bound protein partner by means of luminescent oxygen channeling. In a proof-of-principle study we demonstrate that the format is feasible for efficient identification of small drug-like molecules that bind to a protein target, in this case to the Sec7 domain of cytohesin. We extended the approach to a new cytohesin-specific single chain DNA aptamer, C10.41, which exhibits a similar binding behavior to cytohesins but has the advantage of being more stable and easier to synthesize and to modify than the RNA-aptamer M69. The results obtained with both aptamers indicate the general suitability of the aptamer-displacement assay based on luminescent oxygen channelling (ADLOC) for HTS. We also analyzed the potential for false positive hits and identified from a library of 18,000 drug-like small molecules two compounds as strong singlet-oxygen quenchers. With full automation and the use of commercially available plate readers, we estimate that the ADLOC-based assay described here could be used to screen at least 100,000 compounds per day.
Chemistry 09/2010; 16(36):11100-7. · 5.93 Impact Factor
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Angewandte Chemie International Edition 06/2010; 49(27):4674-7. · 13.45 Impact Factor
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ABSTRACT: In vitro evolution of nucleic acid aptamers is a powerful tool to investigate the structure-function relationship of natural occurring RNA-protein interaction motifs. Otherwise, it also allows the identification of novel RNA-based ligands that can be used to investigate a target's function in its native environment. However, artifacts have been described during in vitro selection procedures hampering the successful enrichment of aptamers. Here we describe a novel observation, namely the enrichment of pan-protein binding RNA sequences. We demonstrate that evolution of specific target binding sequences originating from a pan-protein binding RNA precursor is possible in general. Our data demonstrate that the mutual co-variation of an ancestor molecule can be applied for the evolution of specific target binding RNA sequences. These results might have implications in the context of the RNA world theory, exemplifying a possible evolutionary route towards protein-specific RNA molecules from a common ancestor.
Bioorganic & medicinal chemistry letters 06/2010; 20(12):3793-6. · 2.65 Impact Factor
