Details zur Publikation

Kategorie Textpublikation
Referenztyp Zeitschriften
DOI 10.1371/journal.pone.0134403
Titel (primär) In vitro selection and interaction studies of a DNA aptamer targeting protein A
Autor Stoltenburg, R.; Schubert, T.; Strehlitz, B.
Erscheinungsjahr 2015
Department BOOEK; UBZ
Band/Volume 10
Heft 7
Seite von e0134403
Sprache englisch
UFZ Querschnittsthemen RU1
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.
dauerhafte UFZ-Verlinkung
Stoltenburg, R., Schubert, T., Strehlitz, B. (2015):
In vitro selection and interaction studies of a DNA aptamer targeting protein A
PLOS One 10 (7), e0134403 10.1371/journal.pone.0134403