Electrostatic-mediated Binding of DNA to Lysozyme: Evaluation of Aptamer-based Assays for Highly Positively Charged Targets

Lysozyme is a highly popular protein target for the development of aptamer-based biosensors. Because lysozyme is a polycation and DNA is a polyanion, it is essential to separate the contribution of nonspecific electrostatic interactions from specific aptamer binding. In this study, various factors affecting the binding of DNA and lysozyme, including DNA sequence, DNA length, pH, and salt concentration, were explored using fluorescence polarization. We concluded that direct fluorescence polarization and fluorescence intensity change are unlikely to be directly applicable for aptamer-based biosensors to detect lysozyme because all tested DNA sequences showed binding. These fundamental studies confirm the dominant role of electrostatic binding. We further evaluated three other methods including label-free fluorescent detection using a DNA staining dye, label-free colorimetric detection using gold nanoparticles, and a fluorescent sensor based on the strand displacement reaction. In each case, we focused on a random DNA sequence that is not expected to bind to lysozyme as an aptamer. Of all the methods, only the strand displacement strategy can be potentially used to evaluate aptamer binding, as the other methods all responded to non-aptamer sequences. This study provides valuable insights for assaying aptamer binding to cationic proteins that can exhibit nonspecific attraction to DNA.