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Novel Cap2 structure 5' cap analogue and preparation method thereof

A technology of analogs and caps, which is applied in the field of genetic engineering, can solve the problems of high immunogenicity, low mRNA yield, and low capping rate, and achieve the effects of high synthesis efficiency, low immunogenicity, and high capping efficiency

Active Publication Date: 2020-11-17
SHENZHEN RHEGEN BIOTECHNOLOGY CO LTD
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AI Technical Summary

Problems solved by technology

[0004] Based on the shortcomings of the cap structure analogs in the existing mRNA synthesis system, such as low capping rate, low mRNA yield per unit synthesis volume, and high immunogenicity, a new type of Cap2 cap structure analog has been developed, which not only makes up for the above shortcomings in application , and greatly improved protein translation efficiency

Method used

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  • Novel Cap2 structure 5' cap analogue and preparation method thereof
  • Novel Cap2 structure 5' cap analogue and preparation method thereof
  • Novel Cap2 structure 5' cap analogue and preparation method thereof

Examples

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preparation example Construction

[0097] The present invention also provides a preparation method of the novel Cap2 structure 5' cap analog described in the above technical scheme, comprising the following steps:

[0098] 1) Dissolve 2'-O-Methyl-ATP, 2'-O-Methyl-GTP, 2'-O-Methyl-CTP and 2'-O-Methyl-UTP in RNase-free water respectively, and then mix with Phosphohydrolase and 2×Reaction Buffer were mixed and incubated to obtain 2′-O-Methyl-ADP, 2′-O-Methyl-GDP, 2′-O-Methyl-CDP and 2′-O-Methyl-UDP;

[0099] 2) 2'-O-Methyl-ADP, 2'-O-Methyl-GDP, 2'-O-Methyl-CDP and 2'-O-Methyl-UDP obtained in step 1) were dissolved in RNA-free Mixed with 7-Methylguanosine, 7-Methyl-3′-O-Methylguanosine, guanosyltransferase, and 2×Reaction Buffer in enzyme water for incubation to obtain m7G(5′)ppp(5′)(2′OMeA / G / C / U) and 3'-O-Me-m7G(5')ppp(5')(2'OMeA / G / C / U);

[0100] 3) m7G(5')ppp(5')(2'OMeA / G / C / U) and 3'-O-Me-m7G(5')ppp(5')( 2′OMeA / G / C / U) were dissolved in RNase-free water, mixed with T4 RNA Ligase1, and then mixed with 2′-O-Meth...

Embodiment 1

[0110] 1. Dissolve 10mmol of 2'-O-Methyl-ATP, 2'-O-Methyl-GTP, 2'-O-Methyl-CTP or 2'-O-Methyl-UTP in RNase-free water, total The volume is 14 μl. Add 1μl (50000U) phosphohydrolase and 15μl 2×ReactionBuffer (50mM Tris-HCl; 5mM KCl; 1mM MgCl 2 ; 1mM DTT; pH 8), incubated at 37°C for 1h to obtain 2′-O-Methyl-ADP, 2′-O-Methyl-GDP, 2′-O-Methyl-CDP and 2′-O-Methyl-UDP .

[0111] 2. Purify the 2'-O-Methyl-ADP, 2'-O-Methyl-GDP, 2'-O-Methyl-CDP and 2'-O-Methyl-UDP obtained above by HPLC and dissolve them in RNA-free Enzyme water, total volume 11 μl.

[0112] 3. Add 10 μl (10mmol) 7-Methylguanosine, 10 μl (10mmol) 7-Methyl-3′-O-Methylguanosine and 1 μl (50000U) guanosine transferase to the solution obtained in step 2, add 22 μl 2×ReactionBuffer (50mM Tris-HCl; 5mM KCl; 1mM MgCl 2 ; 1mM DTT; pH 8), incubated at 37°C for 1h to obtain m7G(5′)ppp(5′)(2′OMeA / G / C / U) or 3′-O-Me-m7G(5′) ppp(5')(2'OMeA / G / C / U).

[0113] 4. The solution obtained in step 3 was purified by HPLC and dissolved ...

Embodiment 2

[0148] The 32 novel Cap2 cap structure analogs prepared in Example 1 were applied to mRNA synthesis to effectively increase the synthesis efficiency of mRNA (4-6 mg / ml).

[0149] experimental method:

[0150] 1. Before synthesizing mRNA, linearize the plasmid with NotI and digest overnight at 4°C.

[0151] 2. DNA template extraction.

[0152] 3. In vitro transcription and synthesis of mRNA, respectively using ARCA, Cleancap and 32 kinds of Cap2 in the present invention as the cap structure, the reaction system is shown in Table 1:

[0153] Table 1 Reaction system

[0154] components Dosage T7 10X Reaction Buffer 2ul T7 ATP Solution(75mM) 2ul T7 CTP Solution(75mM) 2ul T7 GTP Solution(75mM) 2ul T7 UTP Solution(75mM) 2ul Linearized plasmid template <8ul

T7 Enzyme Mix 2ul ARCA / Cleancap / Cap2 2ul Nuclease-free water up to 20ul

[0155] React at 37°C for 6 hours. TURBO DNase digestion for 15 minutes.

[015...

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Abstract

The invention provides a novel Cap2 structure 5' cap analogue and a preparation method thereof, and belongs to the technical field of gene engineering. The molecular formula of the novel Cap2 structure 5' cap analogue is selected from any of m7G (5') ppp (5 ') (2' OMeA) p (2 'OMeG), m7G (5') ppp (5') (2 'OMeG) p (2' OMeG) and the like. The novel Cap2 structure 5' cap analogue provided by the invention has higher synthesis efficiency, higher capping efficiency, lower immunogenicity and higher protein translation efficiency.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a novel Cap2 structure 5' cap analog and a preparation method thereof. Background technique [0002] Translation efficiency and immunogenicity are two key factors for the function of mRNA drugs. Autoimmune proteins such as RIG-I and IFIT recognize abnormally capped mRNAs, reducing the activity and half-life of exogenous mRNAs in cells, making it difficult for mRNA drugs to work in vivo. Therefore, optimizing the mRNA cap structure is crucial for enhancing mRNA bioactivity and reducing immunogenicity. [0003] At present, there are two methods for synthesizing capped mRNA in vitro. The first is to cap the mRNA with vaccinia virus capping enzyme after transcription to produce RNA with Cap 0 or Cap 1. This capping method is very efficient. But the output is unstable and the price is very expensive. In addition, the enzymatic capping method cannot produce C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C07H21/02
CPCC12P19/34C07H21/02C12P19/30C12P19/36C12P19/38
Inventor 张苗苗洪丹胡迅
Owner SHENZHEN RHEGEN BIOTECHNOLOGY CO LTD
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