Novel 5′Cap Analog with Cap2 Structure for mRNA Synthesis
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Solution Overview
Problem
Current methods for synthesizing mRNA with cap structures face challenges such as low capping efficiency, low mRNA yield, and high immunogenicity, particularly with existing Cap analogs like ARCA and Cleancap, which limit the biological activity and translation efficiency of mRNA drugs.
Innovation Solution
A novel 5′Cap analog with a Cap2 structure is developed, along with a method involving specific enzymatic reactions to produce m7G(5′)ppp(2′OMeA/G/C/U) and 3′-O-Me-m7G(5′)ppp(2′OMeA/G/C/U) structures, enhancing synthesis, capping efficiency, and reducing immunogenicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If ARCA Cap analog is used for co-transcriptional capping, then immunogenic Cap 0 structure is produced, but capping efficiency is only 70% and mRNA yield is low (maximum 1.5 mg RNA per ml)
Solution Approach 1:
The patent changes the chemical structure parameters of the Cap analog by introducing a Cap2 structure with 2'-O-methyl modifications at specific positions (positions 2 and/or 3). This structural modification transforms the Cap analog from ARCA (Cap 0) or Cleancap (Cap 1) to a Cap2 structure, which simultaneously improves capping efficiency to over 90% and increases mRNA yield to 4-6 mg per ml, while reducing immunogenicity.
2Reliability
If enzymatic capping method is used after mRNA transcription, then Cap 0 or Cap 1 structure is produced, but the method is expensive and yield is unstable
Solution Approach 1:
The patent applies preliminary action by incorporating the Cap2 structure analog directly into the transcription reaction system before transcription begins. The Cap analog is pre-designed with the Cap2 structure containing 2'-O-methyl modifications, allowing it to be incorporated co-transcriptionally. This eliminates the need for subsequent enzymatic capping steps, stabilizes yield, and reduces costs.
3Productivity
If existing Cap analogs are used, then mRNA can be synthesized, but immunogenicity is high which reduces intracellular activity and half-life of exogenous mRNAs
Solution Approach 1:
The patent applies local quality by introducing 2'-O-methyl modifications at specific local positions (positions 2 and/or 3) of the Cap structure, while maintaining the essential Cap structure for recognition. This localized modification reduces immunogenicity detected by RIG-I and IFIT proteins while preserving translation efficiency through eIF4E recognition, thereby improving intracellular activity and half-life without sacrificing mRNA synthesis capability.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The novel Cap2 structure exhibits higher synthetic and capping efficiency, lower immunogenicity, and improved protein translation efficiency compared to existing Cap analogs like ARCA and Cleancap, significantly increasing mRNA yield and translation efficacy.
Implementation Method 1
2'-O-Methyl-ATP, 2'-O-Methyl-GTP, 2'-O-Methyl-CTP and 2'-O-Methyl-UTP were dissolved in RNase-free water respectively, mixed with phosphate hydrolase and 2×Reaction Buffer respectively, and followed by incubation to obtain 2'-O-Methyl-ADP, 2'-O-Methyl-GDP, 2'-O-Methyl-CDP and 2'-O-Methyl-UDP
Implementation Method 2
The 2'-O-Methyl-ADP, 2'-O-Methyl-GDP, 2'-O-Methyl-CDP and 2'-O-Methyl-UDP obtained from the step 1) were dissolved in RNase-free water, mixed with 7-Methylguanosine, 7-Methyl-3'-O Methylguanosine, guanosyltransferase, and 2×Reaction Buffer respectively, and followed by 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)
Implementation Method 3
The m7G(5′)ppp(5′)(2′OMeA/G/C/U) and 3'-O-Me-m7G(5′)ppp(5′)(2′OMeA/G/C/U) obtained from the step 2) were dissolved in RNase-free water respectively, mixed with T4 RNALigase1 and then mixed with 2'-O-Methyl-ATP, 2'-O-Methyl-GTP, 2'-O-Methyl-CTP and 2'-O-Methyl-UTP respectively, followed by mixing with 2×T4 RNA Ligase Reaction Buffer and incubation to obtain the novel 5′Cap analog having a Cap2 structure
Data Source
AI summary
A 5′Cap analog having a Cap2 structure and a preparation method therefor. The molecular formula of the 5′ Cap analog having a Cap2 structure is selected from any one of m7G(5′)ppp(5′)(2′OMeA)p(2′OMeG), m7G(5′)ppp(5′)(2′OMeG)p(2′OMeG), etc. The 5′Cap analog having a Cap2 structure has higher synthesis efficiency, higher capping efficiency, lower immunogenicity, and higher protein translation efficiency.