Part:BBa_K3960017
circRNA scaffold 2
circRNA with MS2 binding site(BBa_K3960006) and PP7 binding site(BBa_K3960007), spacer between them is 10 nt
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
We are SYSU-CHINA 2021.This year, we utilize circRNA as molecular scaffold to colalize enzymes, through the interaction of RNA aptamers and RNA binding proteins.
Design
We used RNA Designer to generate an RNA sequence which contains MS2 aptamer and PP7 aptamer. The distance between two aptamers is 10nt. Except for the two aptamers, no stemloop structures would form based on the prediction. In other words, it presents like a "round shape" circRNA after ligation. The 2D structure was predicted by RNAfold as follows:
The 3D structure was predicted by the service of Xiaolab:
Construction
We used Thermo Scientific TranscriptAid T7 High Yield Transcription Kit (K0411) to carry out in vitro transcription. Firstly, we designed the primers for DNA template amplification through PCR, which are shown as follow:
F:5’-TAATACGACTCACTATAGGGG-3’ R:5’-CAGGTCCGTTGGTTCGTT-3’
After PCR amplification, we subsequently performed in vitro transcription and the protocol is shown as follows:
The result showed that we obtained linear RNA successfully. Next, we used T4 RNA Ligase I to cyclize the linear RNA above. We used T4 RNA Ligase 1(ssRNA Ligase)(NEB M0204S) for ligation. Protocol is listed below:
This result indicated the failure of cyclization since the RNA was almost degradated by RNase R. The possible reason may be the 5'-triphosphate of liner transcripts, which can't be recognized by T4 RNA ligase. RNA 5'-Polyphosphatase can be used to solve this problem. Due to the limit of time, we directly ordered 5'monophosphate modified RNA oligos from Tsingke.We performed ligation again and we finally got our circular RNA scaffold!
The liner RNA and its cyclization product are shown on the picture. Cyclization product has lower migrating rate than linear one. We used RNase R to test the stability of our circRNA against exo-RNase.
The result indicates that our circRNA is resistant to exonuclease like RNase R, which is more stable than the linear RNA.
Application of split EGFP
As for the proof of our concept, we planed to perform split EGFP experiment, which is an commonly used method to detect protein-protein interaction. If the two EGFP fragments can be dragged to each other through the interaction between RNA aptamer and RNA binding proteins, our concept can be proved. The mechanism is listed below:
EGFP splits into to part: EGFP-N and EGFP-C. EGFP-N is fused with MS2 while EGFP-C is fused with PP7 respectively. There is MS2 aptamer and PP7 aptamer on our circRNA scaffold, which has a 10nt length interval. After the binding of two RBPs, the split EGFP fragments can be dragged closer and form a complete EGFP. Then we can perform FCM(flow cytometry) experiment to detect the brightness. Our FCM experiment included four groups. The positive control was transfected with plasmid which can express EGFP while the negative control was transfected with nothing. Experimental group 1 was transfected with plasmids that can express EGFP-N-MS2 and EGFP-C-PP7. The experimental group 2 was transfected with plasmids that can express EGFP-N-MS2 and EGFP-C-PP7 and the circRNA was also transfected into the cells. The results are shown as follows:
The result shows that the percentage of fluorescent cells of experimental group 2 is 17.17%, which is much larger than negative control(0.52%) and experimental group 1(0.66%). This result indicates that the existence of circRNA can promote the interaction between the two fragements of EGFP, which successfully proves our concept.
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