[Comparative thermodynamic analysis of thrombin interaction with anti-thrombin aptamers and their heterodimeric...

Area of Science:

• Biochemistry
• Molecular Biology
• Oligonucleotide Therapeutics

Background:

• Thrombin is a key enzyme in hemostasis.
• Aptamers are short oligonucleotide sequences that bind specific targets.
• Modulating thrombin activity is a therapeutic strategy.

Purpose of the Study:

• To create and characterize dimeric aptamer constructs for enhanced thrombin binding.
• To investigate the thermodynamic and kinetic properties of thrombin-aptamer interactions.

Main Methods:

• Synthesis of dimeric aptamers linked by a poly-(dT) sequence.
• Binding affinity measurements using the Biacore-3000 optical biosensor.
• Thermodynamic analysis of complex formation via temperature-dependent K(D) measurements.

Main Results:

• Dimeric aptamers exhibited 2-3 fold (homodimeric) and 25-30 fold (heterodimeric) lower K(D) values than monomeric aptamers.
• Enthalpy changes (ΔH) for complex formation were similar between aptamers and heterodimeric constructs.
• Entropy changes (ΔS) were 1.5-2 fold higher for heterodimeric constructs, indicating favorable binding.
• Complex formation and dissociation rates increased with temperature, but dissociation was slower for heterodimeric constructs.

Conclusions:

• Dimeric aptamer constructs significantly enhance binding affinity and stability to thrombin.
• The improved binding is primarily driven by favorable entropic contributions.
• These dimeric aptamers represent promising candidates for anticoagulant therapies.