title: Salt-mediated two-site ligand binding by the cocaine-binding aptamer.
abstract: Multisite ligand binding by proteins is commonly utilized in the regulation of biological systems and exploited in a range of biochemical technologies. Aptamers, although widely utilized in many rationally designed biochemical systems, are rarely capable of multisite ligand binding. The cocaine-binding aptamer is often used for studying and developing sensor and aptamer-based technologies. Here, we use isothermal titration calorimetry (ITC) and NMR spectroscopy to demonstrate that the cocaine-binding aptamer switches from one-site to two-site ligand binding, dependent on NaCl concentration. The high-affinity site functions at all buffer conditions studied, the low-affinity site only at low NaCl concentrations. ITC experiments show the two ligand-binding sites operate independently of one another with different affinities and enthalpies. NMR spectroscopy shows the second binding site is located in stem 2 near the three-way junction. This ability to control ligand binding at the second site by adjusting the concentration of NaCl is rare among aptamers and may prove a useful in biotechnology applications. This work also demonstrates that in vitro selected biomolecules can have functions as complex as those found in nature.
authors.affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario, Canada.
authors.forename: Miguel A D
authors.initials: MAD
authors.lastname: Neves
authors.affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario, Canada.
authors.forename: Sladjana
authors.initials: S
authors.lastname: Slavkovic
authors.affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario, Canada.
authors.forename: Zachary R
authors.initials: ZR
authors.lastname: Churcher
authors.affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario, Canada.
authors.forename: Philip E
authors.initials: PE
authors.lastname: Johnson
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D003042:Cocaine / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D011803:Quinine / Q000737:chemistry / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D012965:Sodium Chloride; D011803:Quinine; D003042:Cocaine