Abstract
Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5′-end also contributes essentially to the aptameric function.
Highlights
The enzyme-linked immunosorbent assay (ELISA) is a fundamental tool of immunological, medical, and biochemical research, and was firstly developed in 1971 as a replacement for the radioimmunoassay (RIA)[1]
Enzyme-Linked OligoNucleotide Assay (ELONA) was performed by immobilisation of native or recombinant Protein A in microtiter plates followed by addition of 3′-biotinylated aptamer PA#2/8 for binding
BANK-C represents the starting random oligonucleotide library from which the aptamer PA#2/8 was selected for binding to Protein A by the FluMag-SELEX process as described previously[21]
Summary
The enzyme-linked immunosorbent assay (ELISA) is a fundamental tool of immunological, medical, and biochemical research, and was firstly developed in 1971 as a replacement for the radioimmunoassay (RIA)[1]. Aptamers are obtained by an iterative in vitro selection and amplification method called SELEX, Systematic Evolution of Ligands by Exponential Enrichment[7,8,9], and they are selected for the highly affine and specific recognition and binding to target molecules like small organic compounds, single peptides and proteins, or complex structures and whole cells[10]. Their functionality is based on a complex three-dimensional structure formed by intramolecular folding in accordance with the primary sequence of the aptamers. Several aptamers have been used for the detection of specific bacterial pathogens including E. coli, Salmonella, Campylobacter, Staphylococcus, or Listeria[24,25]
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