Circular RNA Self-Splicing Intron Circularization
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Solution Overview
Problem
Current methods for producing circular RNA in vitro are limited by high costs and restricted size, with existing enzymatic and chemical methods being inefficient and immunogenic, necessitating an improved approach for efficient and low-immunogenic circular RNA production.
Innovation Solution
A circular RNA precursor comprising a 3' self-splicing intron fragment, a first residual circularizing element, a nucleotide sequence of interest, a second residual circularizing element, and a 5' self-splicing intron fragment, which undergoes self-splicing to generate a circular RNA, utilizing a nucleic acid vector and divalent metal cations for in vitro transcription and purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If chemical methods are used to form circular RNAs, then the circularization can be achieved, but the cost increases and the size of circular RNA molecules is limited
Solution Approach 1:
The patent employs self-splicing introns that catalyze their own excision and circularization without requiring external enzymatic catalysts. The intron sequences themselves perform the circularization function through ribozyme activity, eliminating the need for expensive T4 RNA ligase or other catalytic proteins, thus reducing manufacturing cost while enabling larger circular RNA molecules to be produced
Solution Approach 2:
The patent extracts and utilizes the self-splicing activity of introns to achieve circularization. By separating the circularization function from external enzymatic systems and embedding it within the RNA sequence itself through self-splicing introns, the method eliminates dependency on expensive catalysts while maintaining the ability to produce large circular RNA molecules
2Quantity of substance
If T4 RNA ligase is used to catalyze circularization, then circularization can be achieved, but the size of RNA payload is limited and the method is expensive
Solution Approach 1:
The patent replaces T4 RNA ligase with self-splicing introns that perform circularization autonomously. The intron sequences contain the catalytic ribozyme activity needed for circularization, eliminating the need for external enzymatic catalysts. This enables larger RNA payloads to be circularized without cost constraints associated with enzymatic reagents
Solution Approach 2:
The patent substitutes the protein-based T4 RNA ligase system with an RNA-based self-splicing intron system. This replacement eliminates the need for enzymatic catalysts and allows for larger circular RNA molecules to be produced through ribozyme-mediated circularization, which can accommodate bigger payloads without the size limitations imposed by enzymatic methods
3Adaptability or versatility
If ribozyme catalysis is used, then circularization can be achieved with potential for expressible circular RNAs, but the method complexity increases
Solution Approach 1:
The patent segments the circularization process into discrete functional elements: self-splicing introns that perform catalysis, residual circularizing elements that facilitate circularization, and nucleotide sequences of interest that encode the desired circular RNA. This segmentation allows each component to be optimized independently while simplifying the overall method by using modular, pre-characterized elements
Solution Approach 2:
The patent employs universal self-splicing intron sequences that can be applied to various circular RNA designs. The same basic intron framework can be used to circularize different nucleotide sequences of interest, making the method versatile for producing different types of expressible circular RNAs without requiring entirely new methodologies for each application
4Productivity
If natural Group I intron system is used, then circularization can be achieved, but the immunogenicity increases
Solution Approach 1:
The patent modifies specific local regions of the intron sequences to reduce immunogenicity while preserving circularization function. By carefully selecting and optimizing the nucleotide sequences of the self-splicing introns and residual circularizing elements, the method achieves circularization efficiency comparable to natural systems while minimizing immune recognition and adverse immune responses
Solution Approach 2:
The patent changes the nucleotide sequence parameters of the intron and circularizing elements to reduce immunogenicity. By optimizing sequence composition, length, and structural features, the method maintains circularization efficiency while altering the molecular characteristics that trigger immune responses, thereby reducing immunogenicity without sacrificing productivity
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 enables efficient production of circular RNA with reduced immunogenicity, enhancing circularization efficiency and stability, making it suitable for pharmaceutical applications.
Implementation Method 1
Ribozyme catalysis (e.g., based on Group I introns) is a promising method for the preparation of circular RNAs. The natural Group I intron system can undergo cleavage and ligation reactions to form circular intronic RNAs.
Implementation Method 2
A specific cleavage site conserved sequence located in the 5′ exon E1 is cleaved by the nucleophilic attack of the free 3′ hydroxyl group of guanosine triphosphate, resulting in a naked 3′ hydroxyl group
Implementation Method 3
The exposed 3′ hydroxyl group at the 5′ end of the intron attacks the conserved sequence between the 3′ end of the intron and the exon E2, the exon E2 is excised, and the intron undergoes a circularizing reaction
Data Source
AI summary
The present invention relates to the field of biomedicine, in particular, to an improved circular RNA and a preparation method thereof, wherein the improved circular RNA has high generation efficiency and reduced immunogenicity. The present invention also relates to a vector for the preparation of the improved circular RNA, and the use of the improved circular RNA.