title: In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.
abstract: 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.
authors.affiliation: UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle, Germany.
authors.forename: Regina
authors.initials: R
authors.lastname: Stoltenburg
authors.affiliation: 2bind GmbH, Regensburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schubert
authors.affiliation: UFZ-Helmholtz Centre for Environmental Research, Department Environmental and Biotechnology Centre, Leipzig, Germany.
authors.forename: Beate
authors.initials: B
authors.lastname: Strehlitz
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D052156:SELEX Aptamer Technique; D012689:Sequence Homology, Nucleic Acid; D013205:Staphylococcal Protein A / Q000235:genetics* / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D020349:Surface Plasmon Resonance
chemical_list: D052157:Aptamers, Nucleotide; D004269:DNA, Bacterial; D013205:Staphylococcal Protein A