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.

A comprehensive review on staphylococcal protein A (SpA): Its production and applications

“…Rapid and low-cost detection technique for diagnosis of pathogen S. aureus in different environments is critical for infectious diseases control [80][81][82] Using SpA-targeting aptamers conjugated to a porous silicon (PSi) nanostructure to specifically quantify SpA in a range of 2-50 µM in less than 2 H [80] Using carboxyl graphene as the carrier of IgG to develop electrochemiluminescent (ECL) biosensor to detect S. aureus because of specific reaction between SpA and IgG molecules [83] been made to improve the alkaline stability of the IgG-binding domains of the SpA using genetic engineering methods. The C domain has been shown to be fairly resistant to alkaline conditions compared with the other domains, probably due to the presence of Thr-23 instead of the Asn-23 in other domains [78].…”

G-quadruplex-based aptamers against protein targets in therapy and diagnostics

“…Recently, the possibility to detect bacterial pathogens by a G4forming aptamer, selected for Protein A by the FluMag-SELEX process [198], was evaluated in an integrated assay called ELONA (Enzyme-Linked OligoNucleotide Assay) [199], for the recognition of intact bacterial cells of Staphylococcus aureus presenting Protein A on their surface (Fig. 13).…”

Oligonucleotide aptamers: promising and powerful diagnostic and therapeutic tools for infectious diseases

“…Therefore, several ssDNA aptamers have been developed against staphylococcal enterotoxins (SEs). To develop potential diagnostic agents, aptamer APT SEB1 for staphylococcal enterotoxin B (SEB), 42 aptamer C10 for S. aureus enterotoxin C1 (SEC1), 43 aptamer R12.06 for α-toxin, 44 aptamer Antibac1 and Antibac2 for a peptidoglycan 45 and aptamer PA#2/8 for protein A 46 were generated for the detection of SEs. Among these aptamers, aptamer R12.06 for αtoxin was utilized in a modified sandwich ELISA and sensitive detection of 200 nM a-toxin in undiluted human serum samples was achieved.…”