DNA Aptamers to Thrombin Exosite I. Structure-Function Relationships and Antithrombotic Effects

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• DNA aptamers targeting thrombin exosite I possess G-quadruplex structures with specific loop configurations.
• These aptamers inhibit thrombin-mediated fibrinogen binding and platelet aggregation, crucial steps in clot formation.
• The RE31 aptamer, previously synthesized, features a G-quadruplex, hinge region, and a duplex region.

Purpose of the Study:

• To evaluate the impact of structural modifications on the stability and antithrombin activity of the RE31 aptamer.
• To investigate the antithrombotic properties of the RE31 aptamer in various in vitro and in vivo models.

Main Methods:

• Synthesis and characterization of RE31 aptamer analogues with altered duplex regions and hinge regions.
• Assessment of aptamer structural stability using melting temperature measurements.
• Evaluation of antithrombin activity through thrombin, prothrombin, and activated partial thromboplastin time assays in human blood plasma.
• In vitro thrombin generation and fibrin clot lysis assays.
• In vivo arterial thrombosis model in animal studies.

Main Results:

• Reducing the duplex region length decreased aptamer stability and antithrombin efficacy.
• Increasing the duplex region length showed minimal impact on stability and activity.
• Hinge region insertions significantly reduced antithrombin activity.
• RE31 demonstrated efficacy in slowing thrombin formation, accelerating fibrin clot lysis, and suppressing arterial thrombosis.

Conclusions:

• Structural integrity of the duplex region is critical for RE31 aptamer's antithrombin function.
• RE31 aptamer exhibits significant antithrombotic potential.
• RE31 is a promising candidate for further development as an antithrombotic agent.