Regina Stoltenburg

Protein Detection with Aptamer Biosensors

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.

Capture-SELEX: Selection of DNA Aptamers for Aminoglycoside Antibiotics

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 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.

The green fluorescent protein targets secretory proteins to the yeast vacuole

The green fluorescent protein (GFP) was used as a marker to study the intracellular transport of vacuolar and secretory proteins in yeast. Therefore, the following gene constructs were expressed in Saccharomyces cerevisiae under control of the GAL1 promoter: GFP N-terminally fused to the yeast secretory invertase (INV-GFP), the plant vacuolar chitinase (CHN-GFP) and its secretory derivative (CHNDeltaVTP-GFP), which did not contain the vacuolar targeting peptide (VTP), both chitinase forms (CHN and CHNDeltaVTP), GFP without any targeting information and two secretory GFP variants with and without the VTP of chitinase (N-GFP-V and N-GFP). Whereas chitinase without VTP is accumulated in the culture medium the other gene products are retained inside the cell up to 48 h of induction. Independently of a known VTP they are transported to the vacuole, so far as they contain a signal peptide for entering the endoplasmic reticulum. This was demonstrated by confocal laser scanning microscopy, immunocytochemical analysis and subcellular fractionation experiments as well. The transport of the GFP fusion proteins is temporary delayed by a transient accumulation in electron-dense structures very likely derived from the ER, because they also contain the ER chaperone Kar2p/Bip. Our results demonstrate that GFP directs secretory proteins without VTP to the yeast vacuole, possibly by the recognition of an unknown vacuolar signal and demonstrates, therefore, a first limitation for the application of GFP as a marker for the secretory pathway in yeast.

The ALEU2 gene--a new component for an Arxula adeninivorans-based expression platform.

The ALEU2 gene, encoding beta-isopropylmalate dehydrogenase, was isolated from the non-conventional yeast Arxula adeninivorans. The isolated gene harbours an open reading frame of 1086 bp, encoding a putative protein of 362 amino acids. The derived protein sequence shares a high degree of homology with other fungal beta-isopropylmalate dehydrogenases thus confirming the identity of the gene. The isolated ALEU2 gene was tested for its suitability to complement the auxotrophy of an A. adeninivorans aleu2 host. For this purpose the plasmid pAL-ALEU2m which contains the ALEU2 gene as a selection marker and the 25S rDNA for targeting was employed in transformation experiments. Transformants harboured a single copy of the heterologous DNA and were found to be mitotically stable. For assessment of heterologous gene expression, two model genes were incorporated into the vector: the GFP gene, encoding intracellular green fluorescent protein, and the HSA gene, encoding the secreted human serum albumin. For expression control, both gene sequences were fused to the constitutive A. adeninivorans-derived TEF1 promoter and the Saccharomyces cerevisiae-derived PHO5 terminator. In the respective recombinant strains the GFP was localised in the cytoplasm, whereas more than 95% of the HSA accumulated in the culture medium. In initial fermentation trials using a 200-ml shake flask, maximal HSA product levels were observed after 96 h of cultivation.

Halotolerance of the yeast Arxula adeninivorans LS3

The non-pathogenic, dimorphic, ascomycetous yeast Arxula adeninivorans LS3 is halotolerant. It can grow in a minimal medium containing up to 20% NaCl. The growth parameters are only weakly influenced by 10% NaCl. However, NaCl in a concentration higher than 10% causes a decrease in the specific growth rate, a longer adaptation phase and a lower cell count in the stationary growth phase. Concentrations of glycerol and trehalose, which differed 100-fold in magnitude in a salt free medium, are also influenced differently by salt. NaCl induces accumulation of intracellular glycerol in exponentially growing cells but a reduced concentration of intracellular trehalose in stationary cells. Transcripts of the genes ARFC3, encoding a component of the replication factor C, and GAA, encoding a secretory glucoamylase, can be detected only in cells cultured in media with NaCl concentrations below 10%. Furthermore, NaCl in high concentration reduces the level of secreted proteins including glucoamylase end invertase.

Post‐translational modifications of the AFET3 gene product—a component of the iron transport system in budding cells and mycelia of the yeast Arxula adeninivorans

The yeast Arxula adeninivorans is characterized by a temperature‐dependent dimorphism. A. adeninivorans grows as budding cells at temperatures up to 42°C, but forms mycelia at higher temperatures. A strong correlation exists between morphological status and iron uptake, achieved by two transport systems that differ in iron affinity. In the presence of high Fe(II) concentrations (>2 µm), budding cells accumulate iron concentrations up to seven‐fold higher than those observed in mycelia, while at low Fe(II) concentrations (<2 µm), both cell types accumulate similar amounts of iron. The copper‐dependent Fe(II) oxidase Afet3p, composed of 615 amino acids, is a component of the high‐affinity iron transport system. This protein shares a high degree of homology with other yeast iron transport proteins, namely Fet3p of Saccharomyces cerevisiae, Cafet3p of Candida albicans and Pfet3p of Pichia pastoris. Expression of the AFET3 gene is found to be strongly dependent on iron concentration but independent of the morphological stage; however, cell morphology was found to influence post‐translational modifications of the gene product. O‐glycosylation was observed in budding cells only, whereas N‐glycosylation occurred in both cell types. The N‐glycosylated 103 kDa glycoprotein matures into the 108.5 kDa form, further characterized by serine phosphorylation. Both N‐glycosylation and phosphorylation occur at low iron concentrations (≤5 µm). The mature Afet3p of 108.5 kDa is uniformly distributed within the plasma membrane in cells of both morphological stages. Copyright

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

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.

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.

Kinetic and Stoichiometric Characterisation of Streptavidin-Binding Aptamers

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.