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General expression framework of artificial circular RNA and application thereof

An artificial and circular technology, applied in the direction of retroRNA viruses, DNA/RNA fragments, applications, etc., can solve the problems of difficult realization of circRNA chemical synthesis technology, single tumor microRNA network regulation ability, and insufficiency

Active Publication Date: 2018-06-15
浙江自贸区锐赛医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the development of circRNA-based drugs must first solve three basic technical problems: First, the current chemical synthesis technology of circRNA is difficult to achieve
Third, although studies have confirmed that there are multiple target sequences in the circRNA structure and have the ability to simultaneously adsorb and regulate multiple microRNAs, a single endogenous circRNA is still insufficient to regulate the network of tumor microRNAs, and researchers need to develop A circRNA with more or stronger target adsorption regulation ability than endogenous circRNA

Method used

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  • General expression framework of artificial circular RNA and application thereof
  • General expression framework of artificial circular RNA and application thereof
  • General expression framework of artificial circular RNA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Construction of Artificial Circular RNA Overexpression Framework Sequence and Scramble Negative Control Sequence Lentiviral Vector

[0058] 1. Design and synthesis of primers

[0059] Primer design with Primer5 software:

[0060] Circ-F: TTAGGCGCGCCTGAGATTACAGGTGTGAGCC

[0061] Circ-R: GCTTTGTTTAAACGGGATTACAGGTGTGAGCTAC

[0062] The primer sequences were synthesized by Shanghai Huada Gene Company.

[0063] 2. PCR amplification of artificial circular RNA overexpression framework sequence and scramble negative control sequence

[0064] Using the artificial circular RNA overexpression framework sequence or scramble negative control sequence DNA obtained by whole gene synthesis as a template, PCR amplifies the target fragment. The amplification system is as follows:

[0065] 10×Buffer

10ul

MgSO 4 (50mM)

1ul

dNTP (10mM)

1.5ul

transStart Fastpfu DNA polymerase (5U / ul)

0.5ul

Circ-F (10uM)

2ul

Circ-R (1...

Embodiment 2

[0097] Example 2: qPCR detection of overexpression effect of artificial circular RNA and scramble negative control circRNA in 293T cells after lentiviral plasmid transfection

[0098] 1. Colon-circ-1qPCR primer design:

[0099] divergent primer-F1: as shown in SEQ ID NO.7.

[0100] divergent primer-R: as shown in SEQ ID NO.8.

[0101] colon-circ-scramble NC-1qPCR primer design:

[0102] divergent primer-F2: as shown in SEQ ID NO.9.

[0103] divergent primer-R: as shown in SEQ ID NO.10.

[0104] Colon-circ-2qPCR primer design:

[0105] divergent primer-F3: as shown in SEQ ID NO.11.

[0106] divergent primer-R: as shown in SEQ ID NO.12.

[0107] colon-circ-scramble NC-2qPCR primer design:

[0108] divergent primer-F4: as shown in SEQ ID NO.13.

[0109] divergent primer-R: as shown in SEQ ID NO.14.

[0110] The above primer sequences were synthesized by Shanghai Huada Gene Company.

[0111] 2. 24 hours before transfection, digest 293T cells in the logarithmic growth pha...

Embodiment 3

[0149] Example 3: Artificial circular RNA and scramble negative control circRNA and negative control lentivirus (empty virus) packaging

[0150] 1. 24 hours before transfection, digest 293T cells in the logarithmic growth phase with trypsin, transfer to 10cm cell culture dish, 37°C, 5% CO 2 Cultured in an incubator. After 24 hours, when the cell density reaches 70%-80%, it can be used for transfection. Cell state is critical for virus packaging, so good cell state and low passage times need to be guaranteed.

[0151] 2. Replace the cell culture medium with serum-free medium before transfection.

[0152] 3. Add the prepared plasmid DNA solutions (lentiviral plasmid 10 μg, helper plasmids pLP1, pLP2, pLP / VSVG each 5 μg) into a sterilized centrifuge tube, mix with the corresponding volume of Opti-MEM, and adjust the total volume to 1.5ml.

[0153] 4. Shake the Lipofectamine 2000 reagent gently, take 60 μl Lipofectamine 2000 reagent and mix it with 1.5ml Opti-MEM in another tu...

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Abstract

The invention relates to a general expression framework of artificial circular RNA and application thereof. The general expression framework of artificial circular RNA has multi-microRNA combination inhibition ability. The artificial circular RNA overexpression framework consists of three parts: an upstream sequence, an intermediate sequence and a downstream sequence. The artificial circular RNA overexpression framework designed by the invention can achieve effective overexpression and efficient cyclization of the intermediate sequence in cells to produce the artificial circular RNA with multi-microRNA combination inhibition ability. The invention also relates to application of the general expression framework of the artificial circular RNA and eukaryotic expression plasmids, lentiviruses,adenoviruses, adeno-associated viruses and retroviruses carrying the framework in development and preparation of gene therapy drugs.

Description

[technical field] [0001] The invention relates to a general expression framework of artificial circular RNA and its application. [Background technique] [0002] At present, the development of anti-tumor drugs mainly focuses on small molecular compounds (targeted drugs), biological macromolecules (polypeptides, proteins, antibodies), nucleic acid drugs or gene therapy drugs, etc. MicroRNA and siRNA in small nucleic acid molecules have the ability to regulate multiple targets. Research in the past two decades has confirmed that a large number of microRNA play an important role in the occurrence and progression of tumors. As a class of endogenous small nucleic acid molecules, microRNAs have clear biological activities and gene regulation mechanisms. Therefore, microRNAs, as an important class of gene therapy drug development targets, have attracted the attention of researchers. [0003] microRNA is a kind of endogenous small RNA with a length of about 20-24 nucleotides, which ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867C12N15/861C12N15/864A61K48/00A61K31/7088A61P35/00
CPCA61K31/7088C12N15/113C12N15/86C12N2310/10C12N2310/531C12N2710/10043C12N2740/15043C12N2750/14143
Inventor 罗卫峰张腾谭圆圆张立红王秀秀
Owner 浙江自贸区锐赛医学检验实验室有限公司
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