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Capped composition as well as preparation method and in-vitro transcription reaction system thereof

An in vitro transcription and reaction system technology, applied in the fields of chemical and biological engineering, can solve the problems of large steric hindrance, low capping efficiency, poor capping enzyme recognition efficiency, etc., to achieve improved capping efficiency, increased yield, and biocompatibility excellent effect

Active Publication Date: 2022-05-27
JIANGSU SYNTHGENE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the system applied to mRNA capping is more complicated
For example, for some special mRNA sequences, the recognition efficiency of capping enzymes is poor, resulting in low capping efficiency; there are also some mRNA sequences with more secondary structures, which will lead to relatively large steric hindrance when capping, and ultimately Will result in lower capping efficiency

Method used

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  • Capped composition as well as preparation method and in-vitro transcription reaction system thereof
  • Capped composition as well as preparation method and in-vitro transcription reaction system thereof
  • Capped composition as well as preparation method and in-vitro transcription reaction system thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] A capping composition combining figure 1 , prepared by the following method:

[0059] I: Compound m7GDP imidazolium salt (2mmol) was dissolved in MnCl containing 2 (2 mmol) in DMF and added to a 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt) in DMF. The reaction was stirred at room temperature. After 24 hours, the reaction was quenched with 10 mL of 0.25 M EDTA solution. The mixture was loaded onto a DEAESephadex column (3 x 50 cm). The product was eluted using a linear gradient of 0-1.0M TEAB eluent. The eluted product with HPLC purity >97% was collected and concentrated to give the capped analog as triethylamine salt form (c) at a concentration of 120 mM;

[0060] II: the product of converting the triethylamine salt form of the capped analog to the free acid form of the same concentration by a cation exchange resin;

[0061] III: dropwise add 0.2 M TRIS base to the product solution to PH=3~3.6, then concentrate and constant volume to obtain the f...

Embodiment 2

[0063] A capping composition combining figure 2 , prepared by the following method:

[0064] I: Compound m7GDP imidazolium salt (2 mmol) was dissolved in MnCl containing 2 (2 mmol) in DMF and added to a 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt) in DMF. The reaction was stirred at room temperature. After 24 hours, the reaction was quenched with 10 mL of 0.25 M EDTA solution. The mixture was loaded onto a DEAESephadex column (3 x 50 cm). The product was eluted using a linear gradient of 0-1.0M TEAB eluent. The eluted product with HPLC purity >97% was collected and concentrated to give the capped analog in the form of a triethylamine salt at a concentration of 120 mM;

[0065] II: converting the triethylamine salt form of the capped analog to a product of the same concentration as the free acid form by a cation exchange resin;

[0066] III: 0.2M TRIS base is added dropwise to the product solution to PH=5~5.8, and the final product is obtained by concen...

Embodiment 3

[0068] A capping composition combining image 3 , prepared by the following method:

[0069] I: Compound m7GDP imidazolium salt (2 mmol) was dissolved in MnCl containing 2 (2 mmol) in DMF and added to a 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt) in DMF. The reaction was stirred at room temperature. After 24 hours, the reaction was quenched with 10 mL of 0.25 M EDTA solution. The mixture was loaded onto a DEAESephadex column (3 x 50 cm). The product was eluted using a linear gradient of 0-1.0M TEAB eluent. The eluted product with HPLC purity >97% was collected and concentrated to give the capped analog in the form of a triethylamine salt at a concentration of 120 mM;

[0070] II: converting the triethylamine salt form (c) of the capped analog to the product of the free acid form at the same concentration by a cation exchange resin;

[0071] III: 0.2M TRIS base is added dropwise to the product solution to PH=6~7.2, and the final product is obtained by c...

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Abstract

The invention discloses a capping composition as well as a preparation method and an in-vitro transcription reaction system thereof, and belongs to chemical and biological engineering, the inventor of the application applies TRIS to a biological enzyme catalysis system, the composition is mainly composed of a salt solution compounded by a capping analogue and TRIS according to a molar ratio of 1: (3-7), and the TRIS has relatively good biocompatibility, so that the TRIS can be applied to a biological enzyme catalysis system. When some special mRNA sequences are transcribed, the biocompatibility of the TRIS and the mRNA is more excellent, the in-vitro transcription efficiency of the mRNA can be improved, and the capping efficiency of the mRNA can be obviously improved.

Description

technical field [0001] The invention relates to the fields of chemical and biological engineering, in particular to a capping composition, a preparation method and an in vitro transcription reaction system. Background technique [0002] Synthesis of mRNA by in vitro transcription has become an important tool for introducing foreign genetic diseases to express proteins, and is widely used in the treatment and prevention of diseases. In the process of in vitro transcription and synthesis of mRNA, the process of chemical capping of mRNA is an extremely critical step. [0003] However, the system applied to mRNA capping is more complicated. For example, for some special mRNA sequences, the recognition efficiency of capping enzymes is poor, resulting in low capping efficiency; some mRNA sequences have more secondary structures, which will lead to relatively large steric hindrance when capping, and ultimately It will lead to lower capping efficiency. [0004] Therefore, there i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34
CPCC12P19/34
Inventor 黄磊童坤肖潇
Owner JIANGSU SYNTHGENE BIOTECHNOLOGY CO LTD
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