Part:BBa_K4636015

Splint_1802_14

Main

In our research plan, we aim to detect circular RNA (circRNA). However, due to the unavailability of clinical specimen samples currently, we have to synthesize circRNA in our laboratory for testing the feasibility of our detection method. During the synthesis of circRNA, we encountered a challenge because the single-stranded RNA fragment we intended to circularize was relatively long, approximately 300 base pairs. As a result, the efficiency of circularization was expected to be quite low. We considered that the 5' and 3' ends of the RNA might not spontaneously connect together and form a circular structure. To address this issue, we referred to a previously published paper[1] and decided to employ a DNA splint to facilitate the circularization of the RNA. DNA splint can be complementary to the 5’ end and 3’ end of RNA, and then can be circularized under the treatment of T4 RNA ligase 2.[2][3] The working principle of this approach is illustrated in Figure 1.

Figure 1. Schematic diagram of the circularization process using a splint.

Here is the structure of Splint_1802_14. To increase the circularization efficiency, it's important to avoid the formation of secondary structures.

Figure 2. Splint structure predicted by RNAfold web server [4]

Design

For optimal efficiency in circularizing our target RNA, we have considered various design factors based on references[1]. Here are the design considerations:
(1) Keep the GC content below 50% to facilitate the dissociation between DNA-RNA hybrid.
(2) Aim for Tm value around 37°C.
(3) Ensure that the splint is no longer than 20 base pairs, as longer splints may result in a lower combination rate.
(4) To prevent non-specific binding, the splint should not be smaller than 10 base pairs.
(5) The entropy predicted by RNAfold should be close to 1.
(6) Ensure that there are no secondary structures in the splint.

Experiment

Since the splint is employed to aid in the circularization of our target RNA, we conducted experiments to assess the circularization efficiency of splints with different length. However, in Figure 3, we observed that the band in lane 4 is more sharp than others, indicating that the 16-mer splint may have better circularization efficiency than 18-mer splint.

Figure 3. Comparing IVT product of Insert_0101802 circularization efficiency of splint with different length. lane 1: low range RNA ladder, lane 2: Control, lane 3: Circularization product using 14-mer splint, lane 4: Circularization product using 16-mer splint, lane 5: Circularization product using 18-mer splint

Reference

1. An, R., Li, Q., Fan, Y., Li, J., Pan, X., Komiyama, M., & Liang, X. (2017). Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration. *Nucleic acids research, 45(15), e139. https://doi.org/10.1093/nar/gkx553
2. Chen, X., & Lu, Y. (2021). Circular RNA: Biosynthesis in vitro. Frontiers in bioengineering and biotechnology, 9, 787881. https://doi.org/10.3389/fbioe.2021.787881
3. [https://www.neb.com/en/products/m0239-t4-rna-ligase-2-dsrna-ligase#Protocols, Manuals & Usage](https://www.neb.com/en/products/m0239-t4-rna-ligase-2-dsrna-ligase#Protocols,%20Manuals%20&%20Usage)
4. http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi