Christine Reinemann

Aptamers for pharmaceuticals and their application in environmental analytics.

Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affinity and specificity to their target. This property is used for a multitude of applications, for instance as molecular recognition elements in biosensors and other assays. Biosensor application of aptamers offers the possibility for fast and easy detection of environmental relevant substances. Pharmaceutical residues, deriving from human or animal medical treatment, are found in surface, ground, and drinking water. At least the whole range of frequently administered drugs can be detected in noticeable concentrations. Biosensors and assays based on aptamers as specific recognition elements are very convenient for this application because aptamer development is possible for toxic targets. Commonly used biological receptors for biosensors like enzymes or antibodies are mostly unavailable for the detection of pharmaceuticals. This review describes the research activities of aptamer and sensor developments for pharmaceutical detection, with focus on environmental applications.

Generation and characterization of quinolone-specific DNA aptamers suitable for water monitoring.

Quinolones are antibiotics that are accredited in human and veterinary medicine but are regularly used in high quantities also in industrial livestock farming. Since these compounds are often only incompletely metabolized, significant amounts contaminate the aquatic environment and negatively impact on a variety of different ecosystems. Although there is increasing awareness of problems caused by pharmaceutical pollution, available methods for the detection and elimination of numerous pharmaceutical residues are currently inefficient or expensive. While this also applies to antibiotics that may lead to multi-drug resistance in pathogenic bacteria, aptamer-based technologies potentially offer alternative approaches for sensitive and efficient monitoring of pharmaceutical micropollutants. Using the Capture-SELEX procedure, we here describe the selection of an aptamer pool with enhanced binding qualities for fluoroquinolones, a widely used group of antibiotics in both human and veterinary medicine. The selected aptamers were shown to detect various quinolones with high specificity, while specific binding activities to structurally unrelated drugs were not detectable. The quinolone-specific aptamers bound to ofloxacin, one of the most frequently prescribed fluoroquinolone, with high affinity (KD=0.1-56.9 nM). The functionality of quinolone-specific aptamers in real water samples was demonstrated in local tap water and in effluents of sewage plants. Together, our data suggest that these aptamers may be applicable as molecular receptors in biosensors or as catcher molecules in filter systems for improved monitoring and treatment of polluted water.

Investigations on the Specificity of DNA Aptamers Binding to Ethanolamine

In our previous work, we selected aptamers binding to ethanolamine, one of the smallest molecular aptamer targets so far (Mann, D., Reinemann, C., Stoltenburg, R. and Strehlitz, B. Biochem. Biophys. Res. Commun. 2005, 338, 1928-1934). Two representatives of these aptamers (EA#14.3 and EA#9.4) were analyzed regarding their specificity. Ethanolamine is a very small organic molecule (M(w) = 61.08) with biological, medical, and industrial relevance. Its small size represented a challenge for aptamer development, as ethanolamine only consists of a short carbon chain (2C) and two functional groups (amino and hydroxyl group). Related organic molecules, ethanolamine derivatives, and some amino acids were tested to act as potential binding partners for these aptamers. In this way we were able to determine the exact binding domain within the target. The results revealed that both aptamers bind to various molecules, which contain a freely accessible ethyl- or methylamine group. In contrast to the amino group (in a primary, secondary, or tertiary amine) the hydroxyl group was not necessary for the aptamer binding. The aptamers were not able to bind to negatively charged organic molecules, despite containing an ethyl- or methylamine group, nor did they bind to molecules with quaternary amines. The selected ethanolamine binding aptamers are useful for the detection of molecules containing accessible ethyl- or methylamine groups; they can be used as linker elements to immobilize a target molecule of interest on a surface or to purify targets from complex samples.

Identification of the target binding site of ethanolamine-binding aptamers and its exploitation for ethanolamine detection.

Aptamers are promising recognition elements for sensitive and specific detection of small molecules. We have previously selected ssDNA aptamers for ethanolamine, one of the smallest aptamer targets so far. The work presented here focuses on the determination of the binding region within the aptamer structure and its exploitation for the development of an aptamer-based assay for detection of ethanolamine. Sequence analysis of the aptamers resulted in the identification of a G-rich consensus sequence, which was able to fold in a typical two- or three-layered G-quartet structure. Experiments with stepwise truncated variants of the aptamers revealed that the consensus sequence is responsible and sufficient for binding to the target. On the basis of the knowledge of the aptamers binding site, we developed an aptamer-based microarray assay relying on competition between ethanolamine and an oligonucleotide complementary to the consensus sequence. Competitive binding of ethanolamine and fluorescently labeled complementary oligonucleotides resulted in fluorescence intensities dependent on ethanolamine concentration with a limit of detection of 10 pM. This method enables detection of small molecules without any labeling of analytes. The competitive assay could potentially be transferred to other aptamers and thus provides a promising system for aptamer-based detection of diverse small molecules.

S‐layer proteins as an immobilization matrix for aptamers on different sensor surfaces

S‐layer proteins provide a biocompatible environment with different kinds of functional groups, perfect for the sequential coupling of any kind of biofunctional molecule. In addition, their nanostructure and their ability to crystallize on surfaces in a nanometer‐thick monolayer ensure a regular arrangement of these molecules on solid supports. In this work, a thrombin‐binding aptamer and an ofloxacin‐binding aptamer were coupled with different chemical crosslinkers to S‐layer proteins using them for defined immobilization. S‐layer protein monomers and paracrystalline S‐layers were successfully modified with the thrombin‐binding aptamer. However, S‐layer protein monomers were not able to crystallize after aptamer modification and showed no thrombin binding during random surface attachment. In contrast, aptamers linked to an intact S‐layer in suspension or an S‐layer coating were still functional. The modification rate of S‐layers with the thrombin‐binding aptamer was estimated with one aptamer to two unit cells (unit cell = four monomers). Verification of the functionality of both aptamers through target binding after S‐layer‐immobilization on solid supports was proven by laser‐induced fluorescence spectroscopy (LIFS), resonant mirror sensor (IAsys), and quartz crystal microbalance with dissipation monitoring (QCM‐D), respectively. Hence, this study presents S‐layer proteins as an interesting alternative to existing immobilization matrices for recognition biomolecules.