Chronopotentiometric sensing of specific interactions between lysozyme and the DNA aptamer

Abstract

Specific DNA-protein interactions are vital for cellular life maintenance processes, such as transcriptional regulation, chromosome maintenance, replication and DNA repair, and their monitoring gives valuable information on molecular-level organization of those processes. Here, we propose a new method of label-free electrochemical sensing of sequence specific binding between the lysozyme protein and a single stranded DNA aptamer specific for lysozyme (DNA_apta) that exploits the constant current chronopotentiometric stripping (CPS) analysis at modified mercury electrodes. Specific lysozyme-DNA_apta binding was distinguished from nonspecific lysozyme-DNA interactions at thioglycolic acid-modified mercury electrodes, but not at the dithiothreitol-modified or bare mercury electrodes. Stability of the surface-attached lysozyme-DNA_apta layer depended on the stripping current (I_str) intensity, suggesting that the integrity of the layer critically depends on the time of its exposure to negative potentials. Stabilities of different lysozyme-DNA complexes at the negatively polarized electrode surface were tested, and it was shown that structural transitions of the specific lysozyme-DNA_apta complexes occur in the I_str ranges different from those observed for assemblies of lysozyme with DNA sequences capable of only nonspecific lysozyme-DNA interactions. Thus, the CPS allows distinct discrimination between specific and non-specific protein-DNA binding and provides valuable information on stability of the nucleic acid-protein interactions at the polarized interfaces.

Keywords: Aptamer; Catalytic hydrogen evolution; Constant current chronopotentiometric stripping; DNA-protein interactions; Lysozyme; Thiol-modified mercury electrodes.