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

Significance Statement

Affinity reagents are key tools in the field of life sciences, in pharmaceutical and medical research, and therapy. They are of great interest for analytical and diagnostic applications for specific and sensitive detection of biomolecules. DNA aptamers are new affinity molecules matching these specifications. They are short, single-stranded oligonucleotides folding into complex 3D-structures that facilitate highly affine and specific interaction with their target molecules. Aptamers offer outstanding features for practical applications. They are developed by in vitro selection procedure and are produced afterwards by chemical synthesis enabling high availability and reproducibility. Aptamers are small in size, can be easily modified to expand their binding features and to adapt the aptamer to any assay concepts. They possess high storage stability, and regenerability of target binding. Affinity based processes like purification or filtration are further application areas for aptamers.

In this article, a new Protein A – binding aptamer is presented by description of its development and comprehensive characterization of its functionality. Different methods like surface plasmon resonance (SPR) spectroscopy, MicroScale Thermophoresis and bead-based binding assays with fluorescence-labeled aptamer were applied to analyze the binding features. Protein A is known for its ability to bind immunoglobulins and is therefore widely used as affinity matrix in immunological research but also in purification processes throughout the production of commercial/clinical-stage antibodies. This also includes a product testing for leached Protein A as a potential process contaminant. Against this background, the described aptamer has significance for the development of detection assays for Protein A. Moreover, because Protein A is naturally located on the cell surface of Staphylococcus aureus and serves for the binding of the aptamer to the bacterial cells, this aptamer provides the possibility to act as analytical agent for Staphylococcus aureus detection.


Journal Reference

PLoS One. 2015;10(7):e0134403.

Stoltenburg R1, Schubert T2, Strehlitz B3.

Show Affiliations
  1. UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle, Germany.
  2. 2bind GmbH, Regensburg, Germany.
  3. UFZ-Helmholtz Centre for Environmental Research, Department Environmental and Biotechnology Centre, Leipzig, Germany.


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 likeProtein 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.

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Figure legend

Schematic illustration of SPR-based interaction analyses between aptamer and its target Protein A (using Biacore X100). In most of the experiments, biotin-labeled aptamer was immobilized on the streptavidin-modified sensor surface and Protein A was injected as analyte across this surface for interaction with the aptamer. The binding and dissociation profile of a measurement is generated in real-time and recorded in a sensorgram. A typical concentration-dependent binding of Protein A to the aptamer on the sensor surface is shown.

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A. Global Medical Discovery