Clean PIE System for Circular RNA Preparation
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Solution Overview
Problem
Current methods for preparing circular RNA (circRNA) in vitro introduce additional exon sequences, leading to changes in the RNA structure and conformation, triggering cellular immune responses and potential safety hazards in nucleic acid vaccines and gene therapy, with low ligation efficiency and immunogenicity.
Innovation Solution
A recombinant nucleic acid molecule with a novel structure, including intron fragments and truncated translation initiation elements, forms a Clean PIE system that self-splices to produce circRNA without additional exon sequences, improving sequence accuracy and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional in vitro circularization methods (chemical or enzymatic) are used, then circRNA can be produced, but additional exon sequences are introduced leading to changes in RNA structure and conformation
Solution Approach 1:
The translation initiation element is divided into two separate fragments (first fragment and second fragment) that are positioned on opposite sides of the coding sequence. During circularization, these fragments are ligated together to form the complete translation initiation element in the circRNA, avoiding the introduction of additional exon sequences while maintaining proper ribosome binding site functionality.
Solution Approach 2:
The invention extracts and removes the problematic additional exon sequences from the circularization process. By using a specially designed linear nucleic acid molecule where the translation initiation element is fragmented and positioned strategically, the method eliminates the need for extra exon sequences that would otherwise be required for proper circularization, thereby reducing immunogenicity.
2Manufacturing precision
If in vivo circularization is used, then accurate circRNA sequence is achieved, but plasmid integration into genome increases safety risks
Solution Approach 1:
The invention uses a carefully designed linear nucleic acid molecule as an intermediary that contains all necessary elements (fragmented translation initiation element, coding sequence) to produce circRNA with high sequence accuracy through in vitro circularization. This intermediary approach avoids direct plasmid integration into the genome while maintaining the ability to generate accurate circRNA sequences, thereby reducing safety risks associated with in vivo circularization.
3Productivity
If chemical circularization method is used, then circRNA can be formed, but ligation efficiency is low and chemical groups pose safety risks
Solution Approach 1:
The linear nucleic acid molecule is designed with inherent structural features that facilitate self-circularization. The fragmented translation initiation element is positioned such that the 5' end and 3' end of the molecule are brought into proximity, enabling efficient ligation without requiring external chemical catalysts. This self-service approach improves ligation efficiency while avoiding the safety risks associated with chemical groups.
4Manufacturing precision
If protease-catalyzed circularization is used, then circRNA can be produced, but ligation efficiency is low for large fragments and accurate sequences are difficult to obtain
Solution Approach 1:
The invention changes the structural parameters of the nucleic acid molecule by fragmenting the translation initiation element and positioning it strategically across the coding sequence. This parameter change creates a configuration that is more amenable to efficient ligation, thereby improving both ligation efficiency and sequence accuracy simultaneously, overcoming the limitations of protease-catalyzed circularization for large fragments.
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 Clean PIE system enhances the accuracy and stability of circRNA, reducing immunogenicity and safety risks, enabling large-scale industrial production suitable for mRNA infectious disease vaccines, therapeutic mRNA tumor vaccines, and gene therapy.
Implementation Method 1
In the presence of guanine and divalent cations, the intron sequences of Anabaena PIE and T4td PIE form specific structures and are cut down by self-catalysis, thereby circularizing the ribonucleotide sequence in the middle of the intron.
Implementation Method 2
Group I intron self-splicing, Group II intron self-splicing and circularization through some subviral genomes
Data Source
AI summary
The present disclosure relates to a recombinant nucleic acid molecule based on point mutation of translation initiation element and use thereof in the preparation of circular RNA, and in particular to a recombinant nucleic acid molecule for preparing circular RNA, a recombinant expression vector, circular RNA, a composition, a method for preparing circular RNA, a method for expressing a target polypeptide in a cell, a method for preventing or treating a disease, a method for editing a translation initiation element sequence, and a system for editing a translation initiation element sequence. The recombinant nucleic acid molecule provided by the present disclosure provides a novel Clean PIE system for the in vitro preparation of circular RNA, which can avoid introducing additional exon sequences into circular RNA, improve sequence accuracy of circular RNA molecules, reduce changes in a secondary structure of circular RNA, and then reduce immunogenicity of circular RNA, and has a good application prospect in the fields of nucleic acid vaccines, expression of therapeutic proteins, gene therapy, etc.