In the coagulation cascade, a number of proteins are involved such as thrombin, fibrinogen, and tissue factors. Thrombin plays an essential role in the coagulation pathway. Therefore, we designed RNA origami containing thrombin-binding RNA aptamers as anticoagulants.
Two thrombin aptamers that bind specifically at active sites of thrombin called exosite-I and -II were appended to RNA origami
To examine the specificity of RNA origami with thrombin aptamers, we selected tissue factor IXa and X involved in the coagulation cascade and bovine serum albumin as non-specific targets.
The RNA origami-thrombin complexes were tested by gel electrophoresis mobility shift assay.
To test the specificity of binding of RNA thrombin aptamers extended on RNA origami with thrombin
In the complex environment of the coagulation cascade and human plasma, many proteins and small molecules are involved and present (Figure 1). The specific binding of the capture molecule to its target is essential. Thrombin contains two active sites for activation of the coagulation pathway known as exosite-I and -II. In order to inhibit the activity of thrombin, we employ thrombin RNA aptamers that were well studied (1-3). In 2012, Bompiani and colleagues isolated and optimized RNA aptamer called "RNAR9D-14T" that binds prothrombin and thrombin at exosite-I with high affinity (1). The RNAR9D-14T inhibits thrombin generation and thrombin activities such as fibrin clot formation. In 2001, White et al., generated the RNA sequence, Toggle-25, binds exosite-II of thrombin by using the SELEX method (2). The Toggle-25 inhibits plasma clot formation and platelet activation in the coagulation cascade. We selected two potential RNA aptamers. The RNAR9D-14T and Toggle-25 will further be called "apt-1 and apt-2", respectively.
In our project, we designed a functional RNA origami containing two aptamers that bind to thrombin for inhibition of the coagulation process. For testing the specificity of thrombin-binding RNA origami, thrombin, Factor IXa, and Factor Xa that are involved in the coagulation cascade and bovine serum albumin (BSA) were used as test molecules. The binding of the RNA origami-protein complex was characterized by gel electrophoresis mobility shift assay.
RESULTS AND DISCUSSION
We tested the binding complex of RNA thrombin aptamers-containing RNA origami (Th-RNA origami) with thrombin by using gel electrophoresis mobility shift assay. The four designs of RNA origami (one without thrombin aptamers and three designs containing thrombin aptamers) were examined (Figure 2). As we expected, RNA origami without thrombin aptamer named "2HF-RNA-NNNN" is not able to bind with thrombin. All three designs of thrombin-containing RNA origami (2HF-RNA-12NN, 1N2N and 2NN1) incubated with thrombin migrate slower than RNA origami in the absence of thrombin. Moreover, the protein-stained gel shows that the smear pattern bands of thrombin protein appear at the same position of RNA-thrombin complex in the nucleic acid-stained gel. These results indicate that RNA origami with RNA thrombin aptamers bind successfully with thrombin.
We performed specificity tests of the Th-RNA origami with two proteins (Factor IXa and Xa) that involved in the coagulation cascade, and one common protein (bovine serum albumin, BSA). To test specificity, the RNA origami was incubated with protein at 37 C for one hr and characterized by native acrylamide gel electrophoresis. As we expected, the RNA origami without aptamer (2HRF-NNNN) was not able to bind to any of the four proteins (Figure 3). However, all three designs of RNA origami with RNA thrombin aptamers (2HRF-12NN, 1N2N and 2NN1) show specific binding with thrombin as results shown in Figure 4-6.
Additionally, we tested the specific binding of thrombin DNA aptamers decorated on DNA weave tiles with thrombin protein. In our work, 2-helices-DNA weave tile (2HT) was used. For DNA tiles, we extended two aptamers called "Apt-P and Apt-B" on each design that binds with exosite-I and -II of thrombin. DNA tile called "NNNN" does not have any aptamer. For 2HT-DNA-BPNN, aptamer B and P were extended from position 1 and 2 on DNA weave tile (Figure 7). As expected, thrombin binding with 2HT-NNNN was not observed.
We examined the binding of 2HF-RNA, and 2HT-DNA containing two aptamers with thrombin by using gel electrophoresis mobility shift assay. We found that both RNA origami and DNA weave tiles with aptamers bind specifically with thrombin. Additionally, the non-specific binding of RNA origami with non-specific targets (Factor IXa, Xa and BSA) was not found.
MATERIALS AND METHODS
The heat-annealed RNA origami or DNA weave tile (5 pmol) dissolved in 1x annealing buffer was incubated with protein (25 pmol) at 37 °C for 1 hr. The samples were tested by 6% native acrylamide gel electrophoresis in 1X TBE as running buffer at 150 V for 3 hr. The gels were stained with Ethidium bromide for nucleic acid staining and visualized under a UV lamp. Then, the gels were further stained with Coomassie blue for protein staining and imaged with the ProteinSimple instrument.
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
White, R., Rusconi, C., Scardino, E., Wolberg, A., Lawson, J., Hoffman, M. and Sullenger, B., Generation of species cross-reactive aptamers using "toggle" SELEX, Mol Ther. 2001, 4, 567-73.
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.