Difference between revisions of "Part:BBa K3960017"

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	R:5’-CAGGTCCGTTGGTTCGTT-3’		R:5’-CAGGTCCGTTGGTTCGTT-3’
	After PCR amplification, we subsequently perform in vitro transcription as follows:		After PCR amplification, we subsequently perform in vitro transcription as follows:
−	|image [[Image: T7_transcription_in_vitro.png| border | center | 400px]]	+	|image [[Image: Transcript.png| border | center | 400px]]
	|image [[Image: Transcripts.png| border | center | 400px]]		|image [[Image: Transcripts.png| border | center | 400px]]
	<center><font size="1">Figure 3.result of in vitro transcription</font></center>		<center><font size="1">Figure 3.result of in vitro transcription</font></center>

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Revision as of 14:16, 20 October 2021

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

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

Bfore constuction, we first use RNA Designer to generate a RNA sequence, which meets our demangd: containing MS2 aptamer and PP7 aptamer, and the distance between them is 10nt; except for the two aptamers, no stem loop structures should be formed, in other words, it should be more like a "round shape" circRNA after ligation. The 2D structure was predicted by RNAfold as follows:

|image Figure 1.2D structure of circRNA scaffold 2

The 3D structure was predicted by the service of Xiaolab:

|image Figure 2.3D structure of circRNA scaffold 2

Construction

We used Thermo Scientific TranscriptAid T7 High Yield Transcription Kit(K0411) and followed its instruction. First, we have Tsingke Biotechnology synthesized the DNA template which will be used for in vitro transcription. The primers are below: F:5’-TAATACGACTCACTATAGGGG-3’ R:5’-CAGGTCCGTTGGTTCGTT-3’ After PCR amplification, we subsequently perform in vitro transcription as follows:

|image |image Figure 3.result of in vitro transcription

Result shows that we successfully obtain liner RNA. Next, we use T4 RNA Ligase I to cyclize transcript above. The kit we use is the NEB’s T4 RNA Ligase 1 (ssRNA Ligase)(M0204S). Protocol lists below:

|image |image Figure 4.result of ligation

This result indicates false cyclization of liner RNA transcript.The possible reason may be the 5'triphosphate of liner transcripts.We ordered RNA 5'Polyphosphatase to solve this problem, but it has a long delivery time. So we ordered 5'monophosphate modified RNA oligos,which is circRNA scaffold 2(BBa K3960017),and perform ligation again. This time we get our circcular RNA scaffold!

|image Figure 5.successful ligation

The left lane is liner RNA and the right one is cyclization product.Cyclization product has a lower migrating rate than liner ones. To test the stability of our circRNA scaffold against exo-RNase, we performed RNase R experience.However,our circular RNA is sensitive to RNase R, as it put on the instruction of RNase R:it can digest some circRNA which are not resistant to RNase R.

|image Figure 6.RNase R experiment

Lane 1 is liner RNA with RNase R,lane 2 is liner RNA without RNase R,lane 3 is circRNA with RNase R, lane 4 is circRNA without RNase R.After long enough digestion, still some cyclization product exists, indication the successful of our ligation.We will design a more resistant RNA scaffold to RNase R in the future.

Application in split EGFP

For the proof of concept, we planed to perform split EGFP, which is an usual method to detect protein-protein interaction. If we can demonstrate that two EGFP fragments can be dragged to each other through the interaction between RNA aptamer and RNA binding proteins, which are linked to the fragments, then we can say that our concept can be proved. The mechanism lists below:

|image Figure 2.mechanism of split EGFP

EGFP splits into to part: EGFP-N and EGFP-C. EGFP-N is fused to MS2 while EGFP-C is fused to PP7, respectively. There is MS2 aptamer and PP7 aptamer on our circRNA scaffold, which have a 10nt length spacer. After the binding of two RBPs, their fused EGFP fragments can be dragged closer and form a complete EGFP. Then we can perform FCM(flow cytometry) to detect brightness. Our experiment included four groups, which are positive control(only transfected with plasmid which can express EGFP), negative control(transfected with nothing), experimental group(transfected with plasmids that can express EGFP-N-MS2 and EGFP-C-PP7 and circRNA scaffold 2), false positive group(transfected with plasmids that can express EGFP-N-MS2 and EGFP-C-PP7 but no circRNA), respectively. The results are as follows:

|image Figure 3.positive control |image Figure 4.negative control |image Figure 5.co-transfection with circRNA scaffold |image Figure 6.co-transfection without circRNA scaffold

Result shows that the brightness of group4 is between group1 and group2, which conforms to our design. Brightness of group3 is as low as group1, indicating that no false positive effect are interfering our result. All in all, this split EGFP proves that our circRNA scaffold do work.