title: 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 (DNAapta) that exploits the constant current chronopotentiometric stripping (CPS) analysis at modified mercury electrodes. Specific lysozyme-DNAaptabinding 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-DNAaptalayer depended on the stripping current (Istr) 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-DNAaptacomplexes occur in the Istrranges 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.
authors.affiliation: Institute of Biophysics, The Czech Academy of Science, v.v.i., Královopolská 135, 61265 Brno, Czech Republic. Electronic address: ostatna@ibp.cz.
authors.forename: Veronika
authors.initials: V
authors.lastname: Ostatná
authors.affiliation: Institute of Biophysics, The Czech Academy of Science, v.v.i., Královopolská 135, 61265 Brno, Czech Republic.
authors.forename: Veronika
authors.initials: V
authors.lastname: Kasalová-Vargová
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO) and Center for DNA Nanotechnology (CDNA), Science and Technology, Aarhus University, Gustav Wieds Vej 14, DK 8000 Aarhus-C, Denmark.
authors.forename: László
authors.initials: L
authors.lastname: Kékedy-Nagy
authors.affiliation: Institute of Biophysics, The Czech Academy of Science, v.v.i., Královopolská 135, 61265 Brno, Czech Republic.
authors.forename: Hana
authors.initials: H
authors.lastname: Černocká
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO) and Center for DNA Nanotechnology (CDNA), Science and Technology, Aarhus University, Gustav Wieds Vej 14, DK 8000 Aarhus-C, Denmark.
authors.forename: Elena E
authors.initials: EE
authors.lastname: Ferapontova
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D004566:Electrodes; D008628:Mercury / Q000737:chemistry; D009113:Muramidase / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D052157:Aptamers, Nucleotide; D009113:Muramidase; D008628:Mercury
keywords: Aptamer; Catalytic hydrogen evolution; Constant current chronopotentiometric stripping; DNA-protein interactions; Lysozyme; Thiol-modified mercury electrodes