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mRNA and its preparation method and application

A DNA sequence and template technology, applied in DNA preparation, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of poor molecular uniformity, many by-product sequences, and low synthesis efficiency, and achieve strong stability and preparation High efficiency and the effect of improving production efficiency

Active Publication Date: 2021-09-28
GENEO MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problems of low synthesis efficiency, many by-product sequences, poor homogeneity of synthesized molecules, and difficult purification in existing mRNA drug molecule synthesis methods, the present invention provides an mRNA preparation method

Method used

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  • mRNA and its preparation method and application
  • mRNA and its preparation method and application
  • mRNA and its preparation method and application

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Experimental program
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preparation example Construction

[0026] The invention provides a method for preparing mRNA.

[0027] The preparation method of this mRNA comprises the following steps:

[0028] Step S01. Provide DNA transcription template;

[0029] The DNA transcription template is composed of a sense strand and an antisense strand, wherein the sense strand comprises a promoter sequence and a target DNA sequence connected to the promoter sequence, and the promoter sequence does not contain a stop codon, so The target DNA sequence is identical to the target mRNA sequence, and the end of the target DNA sequence is connected with a polyadenylic acid sequence;

[0030] Step S02. Under the action of RNA polymerase, the DNA transcription template is transcribed in vitro, and mRNA is transcribed under the guidance of the promoter sequence.

[0031] The technical scheme of the present invention is explained in detail below:

[0032] In step S01, in the DNA transcription template, the promoter sequence on the sense strand is select...

Embodiment 1

[0061] Embodiment 1 provides a kind of preparation method of green fluorescent protein mRNA (being GFP mRNA), and this preparation method comprises the following steps:

[0062] S11. In the RNase-free PCR tube, add the following components in order:

[0063]

[0064] Note: The DNA template is a linearized GFP plasmid, which was modified on the basis of the pCDNA3.1 plasmid and linearized with endonuclease Pmel; this is the amount of 20 μL reaction system, other reaction systems, each The amounts of ingredients used may be increased or decreased proportionally.

[0065] S12. React at 37°C for 4-6h.

[0066] S13. Heat at 75°C for 10 minutes to inactivate RNA polymerase.

[0067] S14. Take 2 μL of the product and electrophoresis to detect the transcription effect.

[0068] S15. Add 1 μL RNase-free DNase to the in vitro transcription system to remove template DNA (5U / μL).

[0069] S16. React at 37°C for 30 minutes.

[0070] S17. Rehydrate to 100 μL with RNase-free water. ...

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Abstract

The invention relates to the technical field of mRNA molecules, and specifically provides an mRNA and its preparation method and application. The preparation method of the mRNA comprises the following steps: providing a DNA transcription template; the DNA transcription template is composed of a sense strand and an antisense strand, wherein the sense strand comprises a promoter and a target DNA sequence connected to the promoter sequence, and the promoter The subsequence does not contain a stop codon, the target DNA sequence is identical to the target mRNA sequence, and the end of the target DNA sequence is connected with a polyadenylic acid sequence; under the action of RNA polymerase, the DNA transcription template is transcribed in vitro, Under the guidance of the promoter sequence, mRNA is transcribed. The preparation method of the present invention solves the problem of poor molecular homogeneity caused by the increase of by-product sequences during the Poly A tailing reaction of existing mRNA synthesis, and the preparation method also has the advantages of high preparation efficiency and easy purification of products.

Description

technical field [0001] The invention belongs to the technical field of mRNA molecules, and in particular relates to an mRNA and its preparation method and application. Background technique [0002] With the development of nucleic acid technology, gene therapy has increasingly become a hot spot in the pharmaceutical industry. As an important gene therapy, mRNA drugs are increasingly favored by the market. The delivery of mRNA molecules into cells can produce various proteins, including soluble proteins, membrane proteins and intracellular proteins, especially proteins that can be produced by traditional pharmaceutical methods and obtained. Compared with DNA gene therapy, mRNA is a tool for transient expression, which has better biological safety and druggability. With the development and improvement of molecular biology, genetic engineering, and biochemistry, people can synthesize a large number of mRNA drug molecules in vitro, which brings great opportunities for gene ther...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N15/11A61K31/7088
CPCA61K31/7088C12Q1/6844C12Q2525/173C12Q2521/119
Inventor 王刚杨雨亭
Owner GENEO MEDICINE CO LTD
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