Solid-Phase mRNA Transcription to Suppress dsRNA Formation
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Solution Overview
Problem
Current methods for reducing double-stranded RNA (dsRNA) in mRNA synthesis during in vitro transcription are complex, costly, and result in low yield and instability, affecting the efficacy and safety of mRNA vaccines.
Innovation Solution
A high-yield preparation method involving the use of a solid-phase medium modified with negatively charged groups during the transcription process to inhibit dsRNA formation, which can be easily separated and reused, maintaining mRNA stability and yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-generated harmful factors
If purification methods (cellulose chromatography, RNase III digestion, HPLC) are used to remove dsRNA after transcription, then dsRNA level is reduced, but mRNA yield decreases and process complexity increases
Solution Approach 1:
The patent applies preliminary action by adding a solid-phase medium to the transcription system before transcription begins. This medium proactively prevents dsRNA formation during the transcription process itself, rather than reacting to remove dsRNA after it has formed. The solid-phase medium binds to and removes dsRNA in real-time during transcription, preventing its accumulation and eliminating the need for subsequent purification steps that would reduce mRNA yield.
2Object-generated harmful factors
If enzymatic hydrolysis with RNase III is used to digest dsRNA, then dsRNA is removed, but mRNA integrity is compromised and additional purification steps are required
Solution Approach 1:
The patent applies the extraction principle by using a solid-phase medium that selectively extracts or binds to dsRNA molecules during the transcription process. The solid-phase medium acts as a selective trap that captures dsRNA while leaving mRNA intact in the solution. This physical extraction method avoids the need for enzymatic digestion that could compromise mRNA integrity, and the solid-phase medium can be easily removed by simple filtration or centrifugation.
3Object-generated harmful factors
If chaotropic agents are added to reduce dsRNA formation, then dsRNA production is inhibited, but protein structure and enzyme activity are affected
Solution Approach 1:
The patent applies the intermediary principle by introducing a solid-phase medium as a mediator between the transcription system and dsRNA. Instead of using chaotropic agents that directly interfere with molecular interactions and affect enzyme activity, the solid-phase medium serves as a physical intermediary that binds to dsRNA molecules. This mediator approach allows dsRNA reduction without affecting the transcription enzyme or other protein components, as the solid-phase medium selectively interacts only with dsRNA through its binding capacity.
4Object-generated harmful factors
If magnesium ion concentration is reduced to below 5 mM, then dsRNA production is reduced, but mRNA yield decreases
Solution Approach 1:
The patent applies the copying principle by using a solid-phase medium that creates a physical copy or model of dsRNA binding. The solid-phase medium provides a surface that replicates or mimics the binding interaction between dsRNA and purification reagents, allowing dsRNA to be captured and removed from the transcription system. This copying approach enables dsRNA reduction without requiring changes to magnesium ion concentration or other transcription parameters that would affect mRNA yield.
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 method enhances mRNA yield and stability while reducing dsRNA, improving transfection efficiency and decreasing immune factor expression, suitable for large-scale production.
Implementation Method 1
a solid-phase medium modified with negatively charged groups during the transcription process to inhibit dsRNA formation
Implementation Method 2
which can be easily separated and reused
Data Source
AI summary
Provided is a high-yield method and kit for preparing mRNA by reducing or inhibiting double-stranded ribonucleic acid (dsRNA) formation during in vitro transcription. The preparation method is to add solid phase media during a transcription process. Compared with the existing technology, the present invention has the following advantages: according to the high-yield method and kit for preparing mRNA, different types of negatively charged solid phase media are added during the in vitro transcription, reducing the production of dsRNA by interface regulation, and improving the yield and stability of mRNA; in addition, the transfection efficiency of the mRNA prepared by solid phase regulation is improved, and the expression of immune factors is reduced. The solid phase media used in the method and kit are insoluble in water and do not contaminate the transcription system; after the transcription is completed, the solid phase media can be easily separated, and the operation is simple; after proper treatment, the solid phase media can be reused, thus the method and kit have low costs and can be easily scaled up to industrial-scale production.