Structure and dynamics of the dopamine and ochratoxin A aptamer complexes
Cameron D. Mackereth
Professor, ARNA Laboratory
University of Bordeaux
Wednesday, July 23, 2025
10:30 a.m.
In-person: C2-361
Abstract: DNA-based aptamers specific for small molecules provide scaffolds toward diagnostic and therapeutic applications. Despite a growing diversity of the small molecule-binding DNA aptamers, there remains limited molecular knowledge of their binding mechanisms as few have been characterized at the atomic level. Atomic information provides a key aspect in revealing the mode of recognition by aptamers, and can allow for strategic modification of the design to alter aspects of the ligand-bound complexes. For the first aptamer complex, we have used NMR spectroscopy to obtain structural details of RKEC1, a shortened version of a DNA aptamer previously reported to bind dopamine. We find that upon binding dopamine, RKEC1 forms a compact structure that surprisingly lacks any Watson-Crick duplex regions, and in fact also lacks G-quadruplex regions which often occur in DNA aptamers. We found it necessary to follow a base-pair annotation used for the usually more diverse RNA structures, and we also find a correspondingly wide range of intra- and intermolecular hydrogen bonds throughout the complex. A series of dopamine analogues were used to probe the ligand binding site, and the observed changes in affinity for the analogues are explained by the atomic details. Furthermore, the structure of the ligand-bound aptamer enabled us to optimize the design for an electronic aptamer-based biosensor using RKEC1. In a broader sense, the unexpected DNA structure highlights the need for more aptamer structures to better understand the mechanism used by DNA aptamers to bind small molecules. Conformational dynamics is also a critical component for the free and bound DNA aptamers, and we have looked into this property based on the NMR data and also by molecular dynamics. To highlight the importance of cation interactions, we have also looked at the structure of the ochratoxin A aptamer by using NMR spectroscopy in the presence of cations or cobalt hexammine. To further investigate the role of calcium binding, we have performed native mass spectrometry to capture the various ligand plus ion complexes that show an absolute requirement for calcium in the formation of the complex under the initial conditions used to generate the aptamer.
Dr. Cameron Mackereth did his PhD at UBC, following a Co-op Biochemistry degree at the University of Waterloo. During a postdoc at the European Molecular Biology Laboratory in Heidelberg, Germany, Dr. Mackereth focused on using NMR spectroscopy to study the molecular details of protein-RNA complexes. Since 2007, his group at the University of Bordeaux has continued to look at the recognition and structure of nucleic acids, including recent work with small molecule-binding aptamer complexes. Dr. Mackereth is also affiliated to the French National Institute of Health and Medical Research (INSERM) where he is a Research Director.