Oligonucleotide libraries - variatio delectat

Famulok, Michael; Jenne, Andreas

doi:10.1016/s1367-5931(98)80004-5

Cited by 66 publications

(39 citation statements)

References 66 publications

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“…Anti-RTA Aptamers Represent a Novel Class of Nucleic Acid Inhibitors-Aptamers have been shown previously to inhibit a variety of protein targets (35,36). In general, though, there was little or no possibility that previous aptamers could be acted on by their targets.…”

Section: Figmentioning

confidence: 99%

In Vitro Selection of RNA Molecules That Inhibit the Activity of Ricin A-chain

Hesselberth

Miller

Robertus

et al. 2000

Journal of Biological Chemistry

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The cytotoxin ricin disables translation by depurinating a conserved site in eukaryotic rRNA. In vitro selection has been used to generate RNA ligands (aptamers) specific for the catalytic ricin A-chain (RTA). The anti-RTA aptamers bear no resemblance to the normal RTA substrate, the sarcin-ricin loop (SRL), and were not depurinated by RTA. An initial 80-nucleotide RNA ligand was minimized to a 31-nucleotide aptamer that contained all sequences and structures necessary for interacting with RTA. This minimal RNA formed high affinity complexes with RTA (K d ‫؍‬ 7.3 nM) which could compete directly with the SRL for binding to RTA. The aptamer inhibited RTA depurination of the SRL and could partially protect translation from RTA inhibition. The IC 50 of the aptamer for RTA in an in vitro translation assay is 100 nM, roughly 3 orders of magnitude lower than a small molecule inhibitor of ricin, pteroic acid, and 2 orders of magnitude lower than the best known RNA inhibitor. The novel anti-RTA aptamers may find application as diagnostic reagents for a potential biological warfare agent and hold promise as scaffolds for the development of strong ricin inhibitors.Ribosome-inactivating proteins inhibit protein synthesis by disabling the translation machinery. Class I ribosome-inactivating proteins have a catalytic A-chain that recognizes and depurinates a universally conserved adenosine found in a GAGA tetraloop in the sarcin-ricin loop (SRL) 1 of eukaryotic 23-28 S rRNA (1, 2). Class II ribosome-inactivating proteins have an additional lectin B-chain that is required for cell surface attachment and subsequent endocytosis of the toxin domain (3).Ricin, from the castor bean plant Ricinus communis, is a class II ribosome-inactivating protein that has the potential of being used as a weapon in a biological attack (4, 5). Once inside a cell, ricin is extremely toxic; one resident molecule is sufficient to kill the cell, and the enzyme has been reported to inactivate 1777 ribosomes min Ϫ1 (2). Aerosolized ricin causes severe respiratory distress and is lethal when injected intravenously at a level of only 3-5 g/kg (6). Ricin has so far proved to be a relatively inefficient biological weapon but is prepared easily even by Third World nations and terrorist groups. Therefore, facile detection of the toxin and the development of antidotes remain priorities (6, 7).In vitro selection is a powerful molecular tool that can be used to generate ligands for a wide variety of targets, including both nucleic acid and non-nucleic acid-binding proteins (8, 9). Aptamers can be engineered readily to function as either therapeutics or sensors. For instance, RNA aptamers that bind to human immunodeficiency virus type I Rev also inhibit viral replication (10). Similarly, RNA aptamers that bind to the ␤ 2 integrin leukocyte function-associated antigen 1 specifically inhibit a signal transduction pathway when expressed in vivo (11). Consequently, aptamers that recognize and potentially inhibit ricin might be useful prophylactic or therapeutic agent...

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Section: Figmentioning

confidence: 99%

In Vitro Selection of RNA Molecules That Inhibit the Activity of Ricin A-chain

Hesselberth

Miller

Robertus

et al. 2000

Journal of Biological Chemistry

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“…Aptamers can bind a wide variety of targets with high specificity and affinity [11,12], often showing affinity and specificity equaling or even surpassing those of antibodies [13]. Their unique chemical characteristics and method of production presents aptamers as excellent biorecognition elements.…”

Section: Introductionmentioning

confidence: 99%

Electronic ‘Off‐On’ Molecular Switch for Rapid Detection of Thrombin

et al. 2006

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An electrochemical molecular beacon aptasensor for fast and sensitive assay of thrombin is developed and characterized. A bifunctional derivative of the thrombin-binding aptamer, 15-base long with a redox-active ferrocene moiety and a pendant hexanethiol linker group at the termini of the aptamer strand was immobilized on Au electrode surface. The electrochemical behavior of the aptasensor was studied using cyclic voltammetry (CV), chronoamperometry (CA), chronopotentiometry (CP), square-wave voltammetry (SWV) and chronopotentiometric stripping analysis (PSA) techniques. Taken together, these experiments support the switch "on" mechanism of the system when the thrombin binds to the immobilized aptamer. The variation of response to the concentration of thrombin, the target protein, was evaluated by square-wave voltammetry and chronopotentiometric stripping analysis in HEPES buffer solution (pH 8.0, 0.01 M). The aptasensor showed a specific response to thrombin in the range 1.0 to 35 nM with a detection limit of subnanomolar level concentrations. The reaction time, reproducibility, specificity and stability of the aptasensor were also studied.

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“…Since the first reports on nucleic acid aptamers more than a decade ago (1)(2)(3), this class of molecules has developed steadily toward general therapeutic (4 -6) and diagnostic (5,(7)(8)(9) applications. The relative ease of aptamer discovery has allowed for selection against a wide range of targets (10) by processes that have recently been automated (11).…”

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confidence: 99%

Construction, Stability, and Activity of Multivalent Circular Anticoagulant Aptamers

Giusto

King

2004

Journal of Biological Chemistry

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Here we describe the design and construction of multivalent circular DNA aptamers. Four aptameric binding motifs directed at blood-borne targets are used as a model set from which larger, multidomain aptamers are constructed in a straightforward manner. Intra-or intermolecular ligation of precursor oligonucleotides provides a stabilizing mechanism against degradation by the predominant exonuclease activity of blood products without the need for post-selection chemical modification. In many cases, circular DNA aptamer half-lives are extended beyond 10 h in serum and plasma, making such constructs viable for therapeutic and diagnostic applications. Duplexes and three-way junctions are used as scaffold architectures around which two, three, or four aptameric motifs can be arranged in a structurally defined manner, giving rise to improved binding characteristics through stability and avidity gains. Circular aptamers targeted against thrombin display improved anticoagulant potency with EC 50 values 2-3-fold better than those of the canonical GS-522 thrombin DNA aptamer. Intrinsic duplex regions provide an opportunity to incorporate additional transcription factor binding motifs, whereas ancillary loops can be used to provide further functionality. These anticoagulant aptamers provide a starting point for merging the principles of DNA nanotechnology with aptameric functions.Since the first reports on nucleic acid aptamers more than a decade ago (1-3), this class of molecules has developed steadily toward general therapeutic (4 -6) and diagnostic (5, 7-9) applications. The relative ease of aptamer discovery has allowed for selection against a wide range of targets (10) by processes that have recently been automated (11). Simultaneous advances in signaling and detection strategies have improved the outlook for aptamer arrays in proteomic and biosensor applications. Whereas the high affinity and specificity of aptamers toward their molecular targets make them well suited for these tasks, the susceptibility of natural nucleic acids to nucleolytic degradation is a serious hurdle for uses in biological fluids. Strategies to improve the nuclease stability of aptamers, as for antisense nucleic acids, have included the use of chemical modifications such as phosphorothioate, locked nucleic acid, 2Ј-Omethyl-and 2Ј-fluoro-nucleotides (12), as well as mirror-image Spiegelmers (13). Of most relevance for potential therapeutic applications, some common nucleic acid stabilization strategies have encountered problems associated with toxic degradation products (14). In addition, modification performed post-selection can alter the subtle binding interactions of the selected natural aptamer (15, 16).Circularization of natural aptamers is an attractive alternative to chemical modification for improving aptamer stability. With the majority of nucleic acid degradation activity arising from plasma exonucleases (17), modification of exposed termini often achieves a sufficient improvement in stability for use in vivo (12), and circular constru...

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Oligonucleotide libraries - variatio delectat

Cited by 66 publications

References 66 publications

In Vitro Selection of RNA Molecules That Inhibit the Activity of Ricin A-chain

In Vitro Selection of RNA Molecules That Inhibit the Activity of Ricin A-chain

Electronic ‘Off‐On’ Molecular Switch for Rapid Detection of Thrombin

Construction, Stability, and Activity of Multivalent Circular Anticoagulant Aptamers

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