Recombinant Nucleic Acid Design for Clean PIE circRNA Production
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Solution Overview
Problem
Existing methods for preparing circular RNAs (circRNAs) in vitro introduce additional exon sequences, leading to structural changes and immune responses, reducing their stability and safety in gene therapy and nucleic acid vaccines.
Innovation Solution
A recombinant nucleic acid molecule is designed with specific intron and truncated coding element arrangements, utilizing ribozyme recognition sites to form circRNAs without additional exon sequences, enhancing sequence accuracy and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If existing methods for preparing circular RNAs in vitro are used, then circularization can be achieved, but additional exon sequences are introduced leading to structural changes and immune responses
Solution Approach 1:
The coding element is divided into two truncated fragments (first truncated fragment and second truncated fragment) that are ligated together to form the complete coding element in the circular RNA. This segmentation allows precise control over the sequence composition, ensuring that only the intended coding sequences are present without additional exon sequences that would trigger immune responses.
Solution Approach 2:
The invention extracts and removes the problematic additional exon sequences from the circularization process. By using a specifically designed recombinant nucleic acid molecule where the coding element is constructed from truncated fragments ligated at defined ribozyme recognition sites, the method eliminates the introduction of unwanted exon sequences while maintaining efficient circularization.
2Stability of the object's composition
If existing methods for preparing circular RNAs in vitro are used, then circularization can be achieved, but structural changes occur reducing stability
Solution Approach 1:
The coding element is pre-configured in the linear recombinant nucleic acid molecule as truncated fragments with defined ribozyme recognition sites at their boundaries. This preliminary arrangement ensures that during in vitro circularization, the ligation occurs precisely at the intended locations, maintaining sequence accuracy and producing a circular RNA with the correct structure for optimal stability.
Solution Approach 2:
The invention replaces traditional chemical ligation methods with ribozyme-mediated circularization. The ribozyme recognition sites enable site-specific self-circularization through RNA catalysis, which occurs under physiological conditions and maintains the native RNA structure, thereby improving both sequence accuracy and stability compared to chemical methods that may cause structural modifications.
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 improves circRNA sequence accuracy, reduces structural changes, and minimizes immunogenicity, increasing stability and safety for clinical applications in mRNA vaccines and gene therapy.
Implementation Method 1
utilizing ribozyme recognition sites to form circRNAs
Data Source
AI summary
The present disclosure relates to a recombinant nucleic acid molecule and an application thereof in preparation of a circular RNA, and in particular, to a recombinant nucleic acid molecule for preparing a circular RNA, a recombinant expression vector, a circular RNA, a composition, a method for preparing a circular RNA, a method for expressing a target polypeptide in a cell, a method for screening a target coding region sequence, a system for screening a target coding region sequence, and a method for screening a ribozyme recognition site sequence. The recombinant nucleic acid molecule provided by the present disclosure provides a Clean PIE system with a novel structure for preparing a circRNA in vitro, which can avoid introducing additional exon sequences into the circular RNA, improve the sequence accuracy of circular RNA molecules, reduce changes in a secondary structure of the circular RNA, and further reduce the immunogenicity of the circular RNA, and has a good application prospects in the fields of nucleic acid vaccines, expression of therapeutic proteins, gene therapy, and the like.