G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA

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.

Development of An Impedimetric Aptasensor for the Detection of Staphylococcus aureus

“…Assuming an aptamer occupies a squared area, the mean distance between two aptamers is 3.15 nm. Due to dimerization and the formation of quadruplexes [ 36 ], the true mean distance of the aptamers is likely >3.15 nm. According to Erickson et al, the partial specific volume of a protein can be calculated from its molecular mass [ 42 ].…”

“…This aptamer development aimed to detect intact bacterial cells of S. aureus via the protein A bound to its cell surface. Binding characteristics of the aptamer to protein A were studied intensively by different methods such as bead-based fluorescent binding assay, surface plasmon resonance (SPR), microscale thermophoresis (MST), and enzyme-linked oligonucleotide assay (ELONA) [ 35 , 36 ].…”

“…In case of the protein A-binding aptamer, a combination of two structural elements is important for its functionality: First, an intact and free 5′-end, folding into an imperfect stem-loop motif, is crucial for binding to protein A. Second, the aptamer folds into a parallel G-quadruplex structure as demonstrated by circular dichroism spectroscopy [ 36 ].…”

Current approaches for RNA-labelling to identify RNA-binding proteins

“…RNA aptamers interact with their respective substrates via electrostatic and hydrophobic interactions along their folded structure (i.e., stem loops, etc.) enabling anchoring (Ni et al 2011;Stoltenburg et al 2016;Sun et al 2016;Mallikaratchy 2017). The process of determining aptamers and the substrates they bind to, is known as the systematic evolution of ligands by exponential enrichment (SELEX) (Mallikaratchy 2017).…”

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).…”