Defining a stem length-dependent binding mechanism for the cocaine-binding aptamer. A combined NMR and calorimetry study.
We have used a combined approach of NMR spectroscopy and isothermal titration calorimetry (ITC) to determine the ligand-binding mechanism employed by a cocaine-binding aptamer. We found that the length of the stem containing the 3' and 5' termini determines the nature of the binding mechanism. When this stem is six base pairs long, the secondary structure of the aptamer is fully folded in the free form and only putative tertiary interactions form with ligand binding. If this stem is shortened by three base pairs, the free form of the aptamer contains little secondary structure, and ligand binding triggers secondary structure formation and folding. This binding mechanism is supported by both NMR spectral changes and the ITC measured heat capacity of binding (ΔC(p)°). For the aptamer with the long stem the ΔC(p)° value is -557 ± 29 cal mol(-1) K(-1) and for the aptamer with the short stem the ΔC(p)° value is -922 ± 51 cal mol(-1) K(-1). Chemical shift perturbation data and the observation of intermolecular NOEs indicate that the three-way junction is the site of ligand binding.
Neves
Miguel A D
MA
Department of Chemistry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3.
Reinstein
Oren
O
Johnson
Philip E
PE
D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D003042:Cocaine / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009690:Nucleic Acid Conformation; D013816:Thermodynamics
D052157:Aptamers, Nucleotide; D003042:Cocaine