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Synthesis method of capping RNA and capping RNA transcription reaction solution

A synthetic method and polynucleotide technology, applied in the field of genetic engineering, can solve the problems of low RNA capping efficiency and achieve the effect of improving capping efficiency and stability

Pending Publication Date: 2022-01-21
SHANGHAI ZHAOWEI TECH DEV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The purpose of the embodiment of the present application is to provide a capping RNA synthesis method and capping RNA transcription reaction solution, which aims to improve the existing problem of low RNA capping efficiency

Method used

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  • Synthesis method of capping RNA and capping RNA transcription reaction solution
  • Synthesis method of capping RNA and capping RNA transcription reaction solution
  • Synthesis method of capping RNA and capping RNA transcription reaction solution

Examples

Experimental program
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Effect test

Embodiment 1

[0194] This embodiment provides a capped RNA transcription reaction solution and a method for synthesizing capped RNA.

[0195] Mix 1 μL 0.8M Tris-HCl pH7.9, 0.92 μL 1M MgCl 2, 1 μL 40 mM spermidine, 0.2 μL 1M DTT, 1 μL 0.2% by volume Triton X-100, 1.5 μL 100 mM ATP, 1.5 μL 100 mM CTP, 1.5 μL 1.5 mM UTP, 0.7 μL 100 mM GTP , 2.8 μL 100 mM ARCA, 1 μL 200 U / μL T7 RNA polymerase, 0.8 μL 1 U / μL inorganic pyrophosphatase, 1 μL 20 U / μL nuclease inhibitor, 1 μL 1ug / μL DNA template, and 4.08 μL nucleic acid-free Enzyme water was mixed to form a 20uL mixed system to obtain capped RNA transcription reaction solution. The capped RNA transcription reaction solution was incubated at 37°C for 2h. Add 1uL 1U / μL DNase I enzyme and carry out the digestion reaction at 37°C for 30min. Add 10.5 μL of 7.5 M LiCl solution, let stand at -20°C for 20 minutes, then centrifuge at 7500 rpm at 4°C for 15 minutes to obtain RNA precipitate, rinse with 75% ethanol twice to obtain RNA product precipitate. ...

Embodiment 2-12、 comparative example 1-15

[0200] Examples 2-12 and Comparative Examples 1-15 respectively provide a capped RNA transcription reaction solution and a capped RNA synthesis method. Please refer to Example 1, the final concentration of ATP, the final concentration of CTP, and the final concentration of UTP in the capped RNA transcription reaction solution of Examples 2-12 and Comparative Examples 1-15 and the capped RNA transcription reaction solution of Example 1. The concentration, the final concentration of GTP, the selection of the capping polynucleotide and the final concentration of the capping polynucleotide are different, see Table 1 for details. The capped RNA transcription reaction solution of Examples 2-12 and Comparative Examples 1-15 is different from the capped RNA transcription reaction solution of Example 1 in the selection of RNA polymerase, the concentration of RNA polymerase and the DNA template. For details, see Table 2; The incubation temperature and incubation time in the capped RNA s...

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Abstract

The invention relates to the technical field of gene engineering, in particular to a synthesis method of capping RNA and a capping RNA transcription reaction solution. The synthesis method of the capping RNA comprises the following steps: mixing raw materials and performing incubating, wherein the raw materials comprise a DNA template, capping polynucleotide, NTPs, RNA polymerase and a solution containing magnesium ions, the DNA template comprises an RNA promoter, a basic group at the -1 site of a template chain of a RNA promoter is cytosine, and The basic groups from the +1 site to the +n site of a template strand of the RNA promoter are respectively complementarily paired with B1 to Bn in capping polynucleotide. According to the application, the basic group at the site 1 of the template strand of the RNA promoter is regulated to be the cytosine, and besides, the basic groups at the site + 1 to the site + n of the template strand of the RNA promoter are respectively regulated to be complementarily paired with the B1 to the Bn in the capping polynucleotide, so that the RNA capping efficiency is remarkably improved, and the stability of the RNA after transcription synthesis is favorably improved.

Description

technical field [0001] The present application relates to the technical field of genetic engineering, in particular, to a method for synthesizing capped RNA and a capped RNA transcription reaction solution. Background technique [0002] At present, the efficiency of enzymatic capping and co-transcriptional capping synthetic RNA is low, which makes the post-transcriptional RNA less stable, unable to resist enzymatic degradation, improve binding affinity with eIF4E, and improve protein translation s efficiency. Contents of the invention [0003] The purpose of the embodiments of the present application is to provide a capping RNA synthesis method and capping RNA transcription reaction solution, which aims to improve the existing problem of low RNA capping efficiency. [0004] The first aspect of the present application provides a method for synthesizing capped RNA, comprising mixing and incubating raw materials. [0005] Starting materials include DNA templates, capped pol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34
CPCC12P19/34
Inventor 方正军陈丽魏民志
Owner SHANGHAI ZHAOWEI TECH DEV
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