Acyclic Nucleoside Primer Enhances mRNA Capping Efficiency
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Solution Overview
Problem
Current cap analogs for in vitro mRNA synthesis suffer from low transcription yield and capping efficiency, and high immunogenicity, which limits the effectiveness of mRNA production for protein expression and therapeutic applications.
Innovation Solution
An oligonucleotide primer with an acyclic nucleoside structure is used for initial capping, incorporating a UNA structure that replaces the traditional five-membered sugar ring, enhancing capping efficiency and reducing immunogenicity by avoiding recognition by the immune system and ribozymes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional cap analogs (mCap, ARCA, CleanCap) are used for in vitro transcription, then mRNA can be synthesized with capped structure, but the transcription yield and capping efficiency remain low and immunogenicity is high
Solution Approach 1:
The patent changes the chemical structure parameter of the cap analog by replacing the ribose sugar ring with an acyclic UNA structure. This structural parameter change eliminates the 3'-OH group that causes reverse transcription and reduces immunogenicity while maintaining capping function, directly resolving the contradiction between low transcription yield and high immunogenicity
Solution Approach 2:
The patent creates a composite cap structure combining m7Gppp with UNA (unlocked nucleic acid) moiety. This composite structure integrates the capping function of m7Gppp with the immunogenicity-reducing properties of acyclic UNA, achieving both high transcription yield and low immunogenicity simultaneously
2Manufacturing precision
If ARCA is used to prevent reverse transcription, then directional integration is improved, but transcription yield remains low requiring additional enzymatic steps
Solution Approach 1:
The patent modifies the chemical structure by removing the ribose ring to create an acyclic structure. This parameter change inherently prevents reverse transcription (improving directional integration) while being naturally incorporated during transcription (improving yield), eliminating the need for additional enzymatic steps
Solution Approach 2:
The acyclic UNA structure is designed to be incorporated co-transcriptionally during the transcription process itself. The structure preliminarily prevents reverse transcription before it can occur, eliminating the need for post-transcriptional enzymatic processing and improving overall productivity
3Manufacturing precision
If CleanCap is used to achieve Cap1 structure directly, then capping efficiency increases to 90%, but immunogenicity remains high and transcription yield is insufficient
Solution Approach 1:
The patent changes the sugar ring parameter from cyclic (ribose) to acyclic (UNA). This parameter change maintains the Cap1 structure's high capping efficiency while simultaneously reducing immunogenicity, as the acyclic structure is not recognized by immune sensors that target cyclic RNA structures
Solution Approach 2:
The patent creates a composite structure combining the Cap1 features with acyclic UNA. This composite achieves high capping efficiency through proper cap structure while the acyclic component reduces immunogenicity, resolving the contradiction between manufacturing precision and harmful factors
Data Source
AI summary
Provided is an oligonucleotide primer with an acyclic nucleoside structure for initial capping, which has a molecular structural formula of m7UNGpppA2′omepG, and has higher mRNA in vitro transcription efficiency, higher capping efficiency, lower immunogenicity and higher protein translation efficiency.