The coagulation cascade involves a series of enzymatic reactions that ultimately produce cross-linked fibrin clots on ruptured vascular and cellular surfaces (1). Anticoagulants disrupt the process of coagulation by blocking key players in the cascade. The regulation of fibrin clot formation by anticoagulants can consequently evade or delay thrombosis, the formation of blood clots, in vital organs such as the heart, lungs, and brain. The life-threatening ramifications of thrombosis include strokes or transient ischemic attacks, heart attacks, deep vein thrombosis, and pulmonary embolisms (2). Anticoagulants are also commonly administered during surgery to prevent clotting at the site of the operation.
Thrombin plays a cardinal role in coagulation by catalyzing the cleavage of fibrinogen, upstream coagulation factors, and platelet receptors. The catalytic active sites and two extended surfaces, termed exosites, of thrombin participate in macromolecular ligand binding and can be blocked using chemical and biological-based molecules to impede coagulation (1). Control of thrombin activity in the coagulation cascade offers benefits for therapeutic, surgical, and clinical applications.
The most commonly prescribed anticoagulant is Warfarin—used previously as a rodenticide in 1945. Warfarin is a vitamin K antagonist that inhibits the synthesis of clotting factors II, VII, IX, and X and endogenous anticoagulant proteins C and S. The body’s sensitivity to vitamin K fluctuations requires strict and timely monitoring of its levels and prompt dosage adjustments. Other forms of anticoagulants include Heparins, Factor Xa Inhibitors, Direct Thrombin Inhibitors, and Fibrobrinolytics (2). Consistent across all current methods of anticoagulation is a narrow therapeutic window for administration from this sensitivity to fluctuations with serious danger of excessive anticoagulation and even tissue hemorrhaging during surgery if dosage is too high. Unfortunately, there is no rapid antidote for chemical-based anticoagulants that can further mediate administration and combat cytotoxic effects.
Nucleic acid therapeutics are an alternative solution to current pharmaceutical anticoagulants (Figure 2 and 3). DNA and RNA aptamers are structures that have been selected for their ability to bind to specific target molecules. We chose aptamers from the literature that were developed to bind to thrombin and disrupt the coagulation cascade. Their advantages include greater biocompatibility, minimization of side effects, a larger therapeutic window, and the ready availability of rapid antidotes. The antidote is a DNA/RNA strand of complementary sequence to the aptamer that can unfold the structure and release thrombin (1). This poses an advantage for clinical applications that require rapid and robust anticoagulation with tight and reversible control. However, these aptamers exhibited poor pharmacokinetics from their small size (below 30 KDa) as they were quickly removed by the kidneys after circulation. Therefore, they required a more concentrated dosage and continuous administration.
We have designed, created, and tested novel functional RNA origami that can bind to thrombin and inhibit coagulation (Figure 4). We appended two aptamers onto RNA origami to increase binding affinity. This allows for a higher local concentration and a lower necessary dosage. The biocompatible nature of RNA and the availability of an antidote can similarly allow for precise treatment with minimal negative side effects. The circulation of RNA origami in the human body is expected to be longer than free aptamers due to its higher molecular weight. Our structures pose a viable version of current prospects for nucleic acid therapeutics and a safer and more controllable solution to current clinical anticoagulants with less side effects.
Ideal Goal: Construct and large-scale produce a novel anticoagulant from functional RNA origami for safer therapeutic applications by using cells as a factory.
Realistic Goal: Design and construct functional RNA origami by in vitro production and test its function as an anticoagulant.
A Checklist of Our Achievements:
Design of the functional RNA origami
Construction of RNA origami
Analysis RNA origami structure
After successfully demonstrating that our functional RNA origami can act as an anticoagulant, our future directions include:
RNA production can occur in vivo by the use of a cell factory. Modifying cells to enables mass production of the RNA origami.
Design RNA origami to compete for binding of viral RNA and proteins, inhibiting viral processes and reducing HIV propagation.
Smart cells can detect external signals from foreign invaders and produce a specific output to combat.
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