Regina Stoltenburg

New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS) to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of Staphylococcus aureus. In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of K_D = 20 ± 1 nM.

In combination with electrochemical impedance spectroscopy, aptamer-based biosensors are a powerful tool for fast analytical devices. Herein, we present an impedimetric aptasensor for the detection of the human pathogen Staphylococcus aureus. The used aptamer targets protein A, a surface bound virulence factor of S. aureus. The thiol-modified protein A-binding aptamer was co-immobilized with 6-mercapto-1-hexanol onto gold electrodes by self-assembly. Optimization of the ratio of aptamer to 6-mercapto-1-hexanol resulted in an average density of 1.01 ± 0.44 × 10¹³ aptamer molecules per cm². As shown with quartz crystal microbalance experiments, the immobilized aptamer retained its functionality to bind recombinant protein A. Our impedimetric biosensor is based on the principle that binding of target molecules to the immobilized aptamer decreases the electron transfer between electrode and ferri-/ferrocyanide in solution, which is measured as an increase of impedance. Microscale thermophoresis measurements showed that addition of the redox probe ferri-/ferrocyanide has no influence on the binding of aptamer and its target. We demonstrated that upon incubation with various concentrations of S. aureus, the charge-transfer resistance increased proportionally. The developed biosensor showed a limit of detection of 10 CFU·mL⁻¹ and results were available within 10 minutes. The biosensor is highly selective, distinguishing non-target bacteria such as Escherichia coli and Staphylococcus epidermidis. This work highlights the immense potential of impedimetric aptasensors for future biosensing applications.

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5′-end also contributes essentially to the aptameric function.

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5’-end including the 5’-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

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.

Small organic molecules are challenging targets for an aptamer selection using the SELEX technology (SELEX—Systematic Evolution of Ligans by EXponential enrichment). Often they are not suitable for immobilization on solid surfaces, which is a common procedure in known aptamer selection methods. The Capture-SELEX procedure allows the selection of DNA aptamers for solute targets. A special SELEX library was constructed with the aim to immobilize this library on magnetic beads or other surfaces. For this purpose a docking sequence was incorporated into the random region of the library enabling hybridization to a complementary oligo fixed on magnetic beads. Oligonucleotides of the library which exhibit high affinity to the target and a secondary structure fitting to the target are released from the beads for binding to the target during the aptamer selection process. The oligonucleotides of these binding complexes were amplified, purified, and immobilized via the docking sequence to the magnetic beads as the starting point of the following selection round. Based on this Capture-SELEX procedure, the successful DNA aptamer selection for the aminoglycoside antibiotic kanamycin A as a small molecule target is described.

Aptamers are oligonucleotide ligands that are selected for high‐affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer–target interactions is available. To this end, streptavidin‐binding aptamers were isolated and characterised by distinct analytical techniques. Binding kinetics of five broadly similar aptamers were determined by surface plasmon resonance (SPR); affinities ranged from 35–375 nM with large differences in association and dissociation rates. Native mass spectrometry showed that streptavidin can accommodate up to two aptamer units. In a 3D model of one aptamer, conserved regions are exposed, strongly suggesting that they directly interact with the biotin‐binding pockets of streptavidin. Mutational studies confirmed both conserved regions to be crucial for binding. An important result is the observation that the most abundant aptamer in our selections is not the tightest binder, emphasising the importance of having insight into the kinetics of complex formation. To find the tightest binder it might be better to perform fewer selection rounds and to focus on post‐selection characterisation, through the use of complementary approaches as described in this study.

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.

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.

Aptamers have been developed for different applications. Their use as new biological recognition elements in biosensors promises progress for fast and easy detection of proteins. This new generation of biosensor (aptasensors) will be more stable and well adapted to the conditions of real samples because of the specific properties of aptamers.