Salt-mediated two-site ligand binding by the cocaine-binding aptamer

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Multisite ligand binding is crucial for biological regulation and biochemical technologies.
• Aptamers, while common in biochemical systems, rarely exhibit multisite ligand binding.
• The cocaine-binding aptamer is a model for developing aptamer-based sensors and technologies.

Purpose of the Study:

• To investigate the ligand-binding behavior of the cocaine-binding aptamer.
• To determine if the cocaine-binding aptamer can exhibit multisite ligand binding.
• To explore the influence of salt concentration on aptamer-ligand interactions.

Main Methods:

• Isothermal titration calorimetry (ITC) was used to quantify binding thermodynamics.
• Nuclear Magnetic Resonance (NMR) spectroscopy was employed to identify binding sites.
• Experiments were conducted under varying sodium chloride (NaCl) concentrations.

Main Results:

• The cocaine-binding aptamer transitions from one-site to two-site binding based on NaCl concentration.
• A high-affinity site is present under all conditions, while a low-affinity site appears at low NaCl concentrations.
• ITC revealed independent operation of the two sites with distinct affinities and enthalpies.
• NMR spectroscopy localized the second binding site to stem 2 near the three-way junction.

Conclusions:

• The cocaine-binding aptamer exhibits tunable, concentration-dependent multisite ligand binding.
• This controllable binding mechanism, influenced by NaCl, is rare in aptamers and valuable for biotechnology.
• In vitro selected aptamers can possess complex functions comparable to natural biomolecules.