Studying Aptamer-Ligand Interactions and Dynamics in the Cocaine-Binding Aptamer through NMR Spectroscopy

Aptamers are oligonucleotide molecules with applications in biosensors, analytical chemistry, and therapeutics. They are often to 10 to 100 nucleotides in length and can be selected to bind a wide range of targets from ions to cells. The cocaine-binding aptamer was selected in 2000 by Milan Stojanovic to bind cocaine but not benzoylecgonine and ecgonine methyl ester, two common cocaine metabolites. Since its development the cocaine-binding aptamer has been found to bind to quinine with an affinity ~50 times stronger than to cocaine. The cocaine-binding aptamer has been used to help test and develop new biosensing systems.

This research hopes to further the understanding of how the cocaine-binding aptamer interacts with its ligands in the hope that these interactions could be used to help understand other aptamer-ligand systems. Using nuclear magnetic resonance (NMR) spectroscopy the base pair dynamics of the cocaine-binding aptamer was investigated as a function of ligand binding. This study was later expanded to include the effects of temperature and buffer composition to determine thermodynamic parameters of base pair dissociation. The results showed a general reduction in dynamics with ligand binding in the aptamer at the ligand-binding site, but little change elsewhere in the aptamer.

The binding of additional ligands to the cocaine-binding aptamer was characterized by NMR spectroscopy. Levamisole is an anti-parasitic worm medication and one of the most common adulterants found in cocaine. Levamisole was found to bind the cocaine-binding aptamer with a weak affinity at the same site as cocaine. The binding of the cocaine-binding aptamer to a set of three-way junction-binding dyes was also investigated. These dyes share a common structure but bind to the aptamer with a range of affinities. These dyes also bound at the same site as cocaine and quinine, with the dyes being able to be displaced by cocaine or quinine depending on their affinity.

Finally, the binding of ochratoxin A to the ochratoxin A-binding aptamer was investigated using NMR. This aptamer had not been previously studied using NMR, was found to fold tightly in response to its ligand, and NMR proton assignments were obtained.