Part:BBa_K1777016
Device to produce circRNA
| DRS-(β-globin SA)-(miR-21 binding site)6-(β-globin SD)-RRS | |
|---|---|
| Function | Cyclized RNA based on sequence |
| Use in | Mammalian cells |
| RFC standard | RFC 10 |
| Backbone | pSB1C3 |
| Submitted by | [http://2015.igem.org/Team:Fudan Fudan 2015] |
The Fudan iGEM team 2015 designed the device with two inverted repeat sequence which can produce circular mir-21 sponge. We nicknamed it "Ouroboros".The ouroboros or uroboros (/jʊərɵˈbɒrəs/; /ɔːˈrɒbɔrəs/, from the Greek οὐροβόρος ὄφις tail-devouring snake) is an ancient symbol depicting a serpent or dragon eating its own tail.
Just like the ouroboros of myth, our Ouroboros cyclized itself by 'biting' its own. This is the plan A of our cyclizing device. We used the inverted repeat sequence, which is inserted into the flanking intron of the circularizing sequence.
We generate long inverted repeat sequence of 400bp and insert this sequence into the flanking intron of the circularizing exon. We choose the inserted sequence which have higher binding affinity to increase the cyclization efficiency. The flanking intron is β-globin intron1, which can improve the expression level of our device RNA, and provide long linker for the back-splicing mechanism.
Figure 1: Construct design. The Ouroboros contains inverted repeat sequences and 6 miR-21 binding sites. Here, DRS means direct repeat sequence and RRS means reverse-complementary repeat sequence.
The prototype of Ouroboros is to generate circRNA sponge to regulate oncomiRs, and this is based on the long half-life time of circRNA. In order to validate this assumption, we designed circRNA degradation experiment to measure circRNA half-life time.In this experiment, we use our device to cyclize the RNA and simulate the prototype in which the Ouroboros are used as research device.
The circularizing exon have 6 mir-21 binding site, which is reported to be the most efficient number for miRNA sponges. The circularizing exon is constructed by overlap PCR, and the binding site is designed to have the highest binding affinity.
In order to build a tool box, we are synthesize the inverted sequence and the circularizing sequence separately, and finally combine these two parts together. Other than mir-21 sponge, we also designed mir-17 sponge circularizing exon. Our toolbox will finally able to regulate different kinds of miRNAs, and facilitate the reseach and health care issues related to miRNA.
To support our testing experiment, we designed a reporter Luciferase device. We inserted two mir-21 binding site to the 3’-UTR of the Luciferase, and it can report the mir-21 concentration by the intense of fluorescent.
Proof of function
We experimentally validate that our device can accelerate the formation of cirRNA. We used pZW1 as the backbone vector firstly and HEK-293T cells to testing this device. We extracted the total RNA of cells and reverse-transcripted all the RNA into cDNA via Reverse Transcription-PCR (RT-PCR). To proof the RNA-circlization, we designed specific primers.
Figure 2: Identification of circRNA via qPCR.Only circRNA can be expanded by PCR with samples in the same mass and be detected by gel electrophoresis. The result shows our device can cyclize the specific RNA sequence efficiently.
Figure 3: Circlization test by PCR. The 400bp and 300bp markers are indicated in lane 1. Control groups (lane 2 and 3) in which cells was transfected by pZW1 vector without insert sequence cannot cyclize RNA, while the target bands with 356bp can be detected in pZW1-circ groups (i.e. experiment group, lane 4, 5, 6 and 7).
In order to test the results precisely and quantitatively, the cDNA was identifued via real-time quantitative PCR (qPCR). Here is results.
Figure 4: Circlization test by qPCR. Here exists significant difference between control groups and pZW1-circ groups.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 462
Illegal XhoI site found at 833 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 804
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