posted on 2024-01-31, 15:35authored byLorenzo Toma, Monica Mattarozzi, Luca Ronda, Valentina Marassi, Andrea Zattoni, Simone Fortunati, Marco Giannetto, Maria Careri
Aptamers
are recognition elements increasingly used for the development
of biosensing strategies, especially in the detection of proteins
or small molecule targets. Lysozyme, which is recognized as an important
biomarker for various diseases and a major allergenic protein found
in egg whites, is one of the main analytical targets of aptamer-based
biosensors. However, since aptamer-based strategies can be prone to
artifacts and data misinterpretation, rigorous strategies for multifaceted
characterization of the aptamer–target interaction are needed.
In this work, a multitechnique approach has been devised to get further
insights into the binding performance of the anti-lysozyme DNA aptamers
commonly used in the literature. To study molecular interactions between
lysozyme and different anti-lysozyme DNA aptamers, measurements based
on a magneto-electrochemical apta-assay, circular dichroism spectroscopy,
fluorescence spectroscopy, and asymmetrical flow field-flow fractionation
were performed. The reliability and versatility of the approach were
proved by investigating a SELEX-selected RNA aptamer reported in the
literature, that acts as a positive control. The results confirmed
that an interaction in the low micromolar range is present in the
investigated binding buffers, and the binding is not associated with
a conformational change of either the protein or the DNA aptamer.
The similar behavior of the anti-lysozyme DNA aptamers compared to
that of randomized sequences and polythymine, used as negative controls,
showed nonsequence-specific interactions. This study demonstrates
that severe testing of aptamers resulting from SELEX selection is
the unique way to push these biorecognition elements toward reliable
and reproducible results in the analytical field.