STABILITY OF RNA ORIGAMI: LONG-TERM STORAGE

BRIEF SUMMARY

  • The 2’-fluoro-modified RNA origami (2HF-RNA-2NN1) is stable for at least 4 weeks of storage at 4°C.

  • The anticoagulant activity of RNA origami remains observable after 4 weeks of storage.

AIM

  • To test the stability and activity loss of RNA origami for long-term storage.

BACKGROUND

In order to move from the lab to clinical uses, it will be necessary that the stability, integrity, and active function of RNA origami as an anticoagulant be maintained during storage. RNA is an unstable molecule that can be degraded in various conditions such as nuclease and alkaline hydrolysis (1-2). For prevention of RNA degradation, low temperatures (-20°C and - 80°C) are a promising approach. However, Ma et al., 2004, reported that RNase activity still remains in frozen conditions at -20°C (3). An alternative solution is freeze- drying storage, but it is a higher-cost procedure and not always practical for clinical uses. In order to maintain the integrity of RNA with practical storage and low cost, we stored the 2’-modified RNA origami in solution in the normal refrigerator (4°C).     

Figure 1  RNA degradation by RNase A and alkaline hydrolysis (1-2). (A) Ribbon structure of RNase A. (B) The putative mechanism of RNA cleavage by RNase A. (C) RNA degradation via alkaline hydrolysis.

Figure 1 RNA degradation by RNase A and alkaline hydrolysis (1-2). (A) Ribbon structure of RNase A. (B) The putative mechanism of RNA cleavage by RNase A. (C) RNA degradation via alkaline hydrolysis.

RESULTS AND DISCUSSION

We examined the stability and activity loss of 2’-modified RNA origami (2HF-RNA-2NN1)  during long-term storage. The RNA origami samples were transcribed and purified at various time points from fresh production to 1 month of storage at 4 °C.  The anticoagulant activity of the functional RNA origami was tested by aPTT assay. The results in Figure 1 show that the RNA origami is stable and maintains its biochemical function for up to 4 weeks.

Figure 2  The anticoagulation activity of 2HF-RNA-2NN1 at fresh production compared with storage at 4°C from 1 week to 4 weeks. (N=3). Error bars are standard deviation.

Figure 2 The anticoagulation activity of 2HF-RNA-2NN1 at fresh production compared with storage at 4°C from 1 week to 4 weeks. (N=3). Error bars are standard deviation.

CONCLUSION

The modified RNA origami is stable at least for 4 weeks of storage at 4°C, normal refrigerator. Moreover, we found that the anticoagulation activity of the modified RNA origami still remains after 4 weeks of storage. The stability of modified RNA origami for long-term storage offers the benefit for clinical uses.

MATERIALS AND METHODS

The 2HF-RNA-2NN1 samples were produced by in vitro transcription at different time points. The transcribed RNA products were purified and self-assembled by heat-annealing. The samples were diluted in 1x annealing buffer to achieve the desired concentration (5 µM) and stored at 4°C prior to coagulation testing.

go to top

REFERENCES

  1. Raines,  R. T., Ribonuclease A, Chem. Rev. 1998, 98, 1045−1065

  2. Li, Y., and Breaker, R. R.,  Kinetics of RNA Degradation by Specific Base Catalysis of Transesterification Involving the 2′-Hydroxyl Group, J. Am. Chem. Soc. 1999, 121, 5364-5372

  3. Ma, S., Huang, Y., and Huystee, R. B. V., Improved plant RNA stability in storage. Anal. Biochem.,2004, 326, 122–124

go to top