NANOWOLVES: PROJECT OVERVIEW

BACKGROUND

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.

Figure 1.  The Coagulation cascade and anticoagulant drugs (3).

Figure 1. The Coagulation cascade and anticoagulant drugs (3).

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.

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PROBLEM

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.

Figure 2.  Nucleic acid-based aptamers for anticoagulation. (A) Thrombin (4). (B-E) Examples of DNA and RNA aptamers developed as anticoagulants due to their ability to bind and inhibit thrombin. (B) RNAR9D-14T and (C) Toggle-25t RNA aptamers bind to exosite-I and exosite-II of thrombin. DNA aptamers called NU172 and HD22 bind to exosite-I and exosite-II of thrombin. (1, 5, 6)

Figure 2. Nucleic acid-based aptamers for anticoagulation. (A) Thrombin (4). (B-E) Examples of DNA and RNA aptamers developed as anticoagulants due to their ability to bind and inhibit thrombin. (B) RNAR9D-14T and (C) Toggle-25t RNA aptamers bind to exosite-I and exosite-II of thrombin. DNA aptamers called NU172 and HD22 bind to exosite-I and exosite-II of thrombin. (1, 5, 6)

Figure 3 The crystal structure of Toggle-25t RNA aptamer bound to exosite-I of thrombin (6)

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.

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OUR SOLUTION

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.

Figure 4 The cartoon represents RNA origami binding to Thrombin.

Note that the illustration is not scientifically accurate and not to scale. The RNA structure shown here is a 2-helix RNA origami without aptamer attached. The 3D model of two aptamers tethering on RNA origami is not possible to create accurately since no crystal structure exists for the exosite-I aptamer.

PROJECT GOALS

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.

Figure 5  Our project goals entailed.

Figure 5 Our project goals entailed.

FUTURE DIRECTIONS

After successfully demonstrating that our functional RNA origami can act as an anticoagulant, our future directions include:

Cell factory

RNA production can occur in vivo by the use of a cell factory. Modifying cells to enables mass production of the RNA origami.

Beyond anticoagulants: HIV Therapy

Design RNA origami to compete for binding of viral RNA and proteins, inhibiting viral processes and reducing HIV propagation.

Smart cells

Smart cells can detect external signals from foreign invaders and produce a specific output to combat.

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REFERENCES

  1. Bompiani, K. M., Monroe, D. M., Church, F. C., and Sullenger B. A., A high affinity, antidote-controllable prothrombin, and thrombin-binding RNA aptamer inhibits thrombin generation and thrombin activity, J Thromb Haemost. 2012, 10, :870-80

  2. Harter, K., Levine, M., & Henderson, S. O. Anticoagulation Drug Therapy: A Review. Western Journal of Emergency Medicine. 2015, 16(1), :11–17.

  3. Wolberg, A. S., Rosendaal, F. R.,Weitz, J. I., Jaffer, I. H., Agnelli, G., Baglin, T., and Mackman, N., Venous Thrombosis, Nat. Rev. Dis. Primers 2015, 1

  4. Deng, B., Lin, Y., Wang, C., Li, F., Wang, Z., Zhang, H., Li, X. F., and Le, X. C.,Aptamer binding assays for proteins: The thrombin example—A review, Anal Chim Acta. 2014, 837, 1-15

  5. Long, S. B., Long, M. B., White, R. R., Sullenger, B. A., Crystal structure of an RNA aptamer bound to thrombin, RNA. 2008, 14, 2504-12.

  6. Rangnekar, A., Nash, J. A., Goodfred, B., Yingling, Y. G., LaBean, T. H.,Design of Potent and Controllable Anticoagulants Using DNA Aptamers and Nanostructures, Molecules 2016, 21, 1-13

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