Designed DNA strands that can be used as antidotes in the anticoagulation assay.
After inhibition of thrombin activity by RNA aptamers, the coagulation activity of thrombin can be reestablished by adding DNA antidotes.
The recovery of thrombin activity is approximately 80%.
To reestablish coagulation activity of thrombin by turning off the RNA aptamers using complementary single-stranded DNA antidotes which hybridize with the aptamer sequences causing them to unfold.
Control over the coagulation cascade offers benefits for surgical and disease applications. Chemical-based anticoagulants have been developed and are frequently used, but due to their small therapeutic window (narrow concentration difference between therapeutic and toxic doses), patients need to be monitored constantly in order to prevent dangerous side-effect such as hemorrhaging and bleeding. An alternative solution is nucleic acid-based anticoagulants that will never induce hemorrhaging and can be turned off by available antidotes.
Antidotes for aptamer-based anticoagulants are short strands of single-stranded DNA with nucleotide sequences complementary to the aptamer sequences. Bompiani, K. M., and colleagues demonstrated how adding ssDNA antidote can reverse inhibition and reactivate of thrombin activity (1). This was also demonstrated with DNA aptamers on DNA tiles (2,3). RNA origami bearing RNA aptamers offers high anticoagulation activity compared with free aptamers, as we demonstrated in coagulation assays. The reversal of thrombin inhibition can be challenging due to the need to interrupt tight thrombin binding and stable folding of the aptamers. Here, we examined the recovery of coagulation activity by adding two antidotes that are complementary counterparts of exosite-1 and -2 binding aptamers. This antidote mechanism may provide great benefit during medical procedures for turning on and off the coagulation cascade.
RESULTS AND DISCUSSION
Picking the most effective design of our RNA origami, 2HF-RNA-2NN1, we designed antidotes made of DNA for both aptamers. After the RNA origami was incubated with plasma and other reagents for an aPTT assay, the antidote was added, and the whole sample was incubated for a further 5 minutes. It was expected that binding to thrombin would be completely reversible.
Data showed that a reversal of activity was possible with the addition of the antidote, as the antidote trial revealed an average clotting time of around 85 seconds in which is 80% recovery (thrombin still 20% inhibited). The incomplete reversal could be due to the antidotes being less effective for strand invasion on one of the two aptamers, or a difference in the concentration of antidote needed to counteract the thrombin binding. In order to achieve full reversal activity, antidotes may need to be improved. For example, a complementary PNA strand may work better than DNA.
The 2HF-RNA-2NN1 origami was shown to be effective at inhibiting and delaying coagulation and to do so at concentrations as low as 0.5 μM. However, another benefit of the RNA origami and RNA aptamer design is easy implementation of antidotes to the aptamers, allowing surgeons to turn off anticoagulation. We demonstrated that the activity of thrombin can be 80% recovered by adding ssDNA antidote. There still exists alternative antidote designs and further study must be completed in order to increase the effectiveness of the antidotes.
MATERIALS AND METHODS
The coagulation test was run in an activated partial thromboplastin time (aPTT) assay with a model ST4 coagulometer (Diagnostica Stago). 50 μL pooled human plasma (George King Bio-Medical) was added in each cuvette, mixed with 50 μL aPTT reagent (TriniClot), and incubated at 37 °C for 5 minutes. Then 13.67 μL of 5 μM RNA origami sample, or buffer, was added and incubated for another 5 minutes at 37°C. After the five minutes, 3.00 μL of DNA antidote was added, and incubated for 5 more minutes at 37°C. To activate clotting, 50 μL CaCl2 solution was added. The final concentration of RNA sample was 0.5 μM. The time to clot was then measured by the coagulometer and recorded.
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
Rangnekar, A., Nash, J. A., Goodfred, B., Yingling, Y. G., and LaBean, T. H., Design of Potent and Controllable Anticoagulants Using DNA Aptamers and Nanostructures, Molecules 2016, 21, 1-13
Rangnekar, A., Zhang, A. M., Li, S. S., Bompiani, K. M., Hansen, M. N., Gothelf, K. V., Sullenger, B. A.,and LaBean, T. H., Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures, Nanomedicine., 2012, 8, 673-681