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A precise type circular RNA expression framework based on an Alu element, a vector and application of the framework and the vector

An alu element and circular technology, which is applied in the field of molecular biology, can solve the problems of inability to successfully overexpress, circularize, and cannot guarantee accuracy and high efficiency, and achieve high accuracy of circularization, accurate ring formation and shearing, and stable effects Effect

Active Publication Date: 2019-05-03
GUANGZHOU GENESEED BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] In addition, in the prior art, some companies have provided the construction of circular RNA overexpression vectors, but they cannot guarantee accurate and high-efficiency circularization, and there are common situations where successful overexpression or circularization errors occur after overexpression. Therefore, designing a highly efficient And accurate circular RNA overexpression technology is imminent

Method used

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  • A precise type circular RNA expression framework based on an Alu element, a vector and application of the framework and the vector
  • A precise type circular RNA expression framework based on an Alu element, a vector and application of the framework and the vector
  • A precise type circular RNA expression framework based on an Alu element, a vector and application of the framework and the vector

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Experimental program
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Effect test

Embodiment 1

[0080] Such as figure 1 As shown, it is a schematic diagram of the Alu element-based circular RNA expression framework of the present invention. Referring to the Alu element information annotated in the RepeatMasker database, based on the conserved sequence of the Alu element of about 40nt, design Alu1 and Alu2 with a length of 300nt, and then design the 200nt intron splicing recognition sequence SRS1 and SRS2 based on the splicing pattern of snRNP. Use RNAfold to predict the secondary structure of the sequence, modify and adjust bases to make Alu1 and Alu2 reverse complementary to form a stable hairpin structure, keep the minimum free energy less than -350.00kcal / mol, adjust and modify bases to make SRS1 and SRS2 form a neck The ring structure, and not paired with the Alu sequence, maintains a minimum free energy greater than -50kcal / mol. Finally, the length of Alu1 is 200nt such as SEQ ID NO:1, the length of Alu2 is 200nt such as SEQ ID NO:5, the length of SRS1 is 153nt suc...

Embodiment 2

[0084] Circularization mediator sequences IS-L18, IS-S10 and IS-Y10 were used to construct the vector of circRNA82002 with pCD5-ciR, and 293T cells were transfected with RT-qPCR to detect the overexpression fold (RNA level), and the PCR product Perform sanger sequencing to verify whether IS-L18, IS-S10 and IS-Y10 can mediate accurate circularization of circRNA82002 overexpression.

[0085] Use primers:

[0086] IS-L18-pCD-ciR-circRNA82002-F:

[0087] ggGGTACCTGAAATATGCTATCTTACAGATAAACCTCTCATAATGAAG;

[0088] IS-L18-pCD-ciR-circRNA82002-R:

[0089] cgGGATCCTCAAGAAAAAATATATTTCACCCTATTAAAGCAGTGCTCAT;

[0090] IS-S10-pCD5-ciR-circRNA82002-F:

[0091] cgGAATTCCATCTTACTTCAGATAAACCTCTCATAATGAAG;

[0092] IS-S10-pCD5-ciR-circRNA82002-R:

[0093] cgGGATCCATGAACTTATACCCTATTAAAAGCAGTGCTCAT;

[0094] IS-Y10-pCD5-ciR-circRNA82002-F:

[0095] cgGAATTCCTAATACTTTTCAGATAAACCTCTCATAATGAAG;

[0096] IS-Y10-pCD5-ciR-circRNA82002-R:

[0097] cgGGATCCAGTTGTTTCTTACCCTATTAAAGCAGTGCTCAT.

...

Embodiment 3

[0137] A total of 326 circular RNA expression vectors for humans, mice and rats were constructed with pCD5-ciR, pLC5-ciR and pK25ssAAV-ciR in the present invention, and RT-qPCR detection was performed based on the RNA level after transient transfection, and the results were as follows Figure 16-17 As shown, the success rate of 50-fold overexpression and accurate cutting into a circle is close to 100%, which is greatly improved compared with the prior art. The following is an example of the detailed results of 4 genes.

[0138] pcDNA3.1(+) CircRNA Mini Vector, pCD-ciR and pCD5-ciR were used to construct the overexpression vectors of circRNA82002, circRNA00284, circRNA05836, and rnocircRNA00978 respectively, and after transfection into 293T cells, RT-qPCR was performed to detect the overexpression fold, and the PCR products were Perform sanger sequencing to verify whether the overexpression can be accurately circularized.

[0139] Use primers:

[0140] pcDNA3.1(+)CircRNA Mini...

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Abstract

The invention relates to a circular RNA expression framework based on an Alu element. The framework is characterized by including one or more Alu elements, one or more restriction enzyme cut sites, one or more splicing recognition sequences, one or more cyclization mediating sequences, a splicing acceptor sequence, a splicing donor sequence and one or more circular RNA sequences. The framework ishigher in universality, can be used for different circular RNAs of different species, can be generally applied for natural and artificially designed circular RNA sequences, and can achieve precise cyclization overexpression for circular RNAs having 200-2500 nt. The framework has high cyclization accuracy, wherein cyclization splicing after overexpression is accurate, precise splicing at AG-GT positions during cyclization is achieved through combination of SRS and IS, and cyclized circular RNAs are free of base addition or deletion. The framework has a high overexpression efficiency, with cyclization overexpression effects being stable, and circular RNAs are significantly overeexpressed by 50 times or tens of thousands of times under the precise of accurate cyclization. The framework is widely applied, is suitable for various demands such as cell transient expression, stable cell expression and animal expression, and is provided with GFP and puromycin resistance gene to enable marking and screening.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to an Alu repeat element-based precise circular RNA expression framework and a vector construction method and application thereof. Background technique [0002] Circular RNAs (circular RNAs, circRNAs) are a class of RNA molecules with a closed circular structure, which widely exist in species such as archaea, nematodes, mice, rats, and humans, and their expression has certain tissue and timing specificity. , and not easily degraded by exonucleases, more stable than linear RNA. A large number of studies have shown that circular RNA can act as miRNA sponge, interact with polymerase II (RNA polymerase II, Pol II) to regulate host transcriptional activity, directly translate proteins and other functional pathways in brain development, Parkinson's disease, Alzheimer's disease and tumors It plays an important role in the occurrence of circRNAs, suggesting the potential of cir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85
Inventor 李自强黄宁宁谢芳梅车水云刘明
Owner GUANGZHOU GENESEED BIOTECH
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