Self-Circularized RNA Structure for Longer Protein Expression
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Solution Overview
Problem
The short half-life of messenger RNA (mRNA) in biological systems limits its effectiveness for therapeutic and engineering applications due to rapid degradation by exonucleases.
Innovation Solution
The method involves transcribing a vector to form a precursor RNA with specific elements arranged to create a thermodynamically stable multiple junction RNA structure, which can form a circular RNA capable of translation and biological activity within cells, using Group I self-splicing introns and internal ribosome entry sites (IRES) to enhance stability and expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If linear mRNA is used for therapeutic applications, then it can be easily synthesized and delivered, but it has a short half-life due to rapid degradation by exonucleases
Solution Approach 1:
The patent merges the 5' and 3' ends of the mRNA molecule to form a circular structure. This circularization process eliminates the free ends that are susceptible to exonuclease degradation, thereby extending the half-life of the mRNA while maintaining its functional properties for protein expression.
Solution Approach 2:
The patent employs self-splicing introns that catalyze their own removal through splicing reactions. This self-service mechanism allows the mRNA to circularize itself without requiring external enzymatic intervention, simplifying the manufacturing process while achieving the desired circular structure.
2Duration of action of stationary object
If circular RNA is formed to extend mRNA half-life, then protein expression duration is prolonged, but the complexity of RNA structure and preparation increases
Solution Approach 1:
The patent segments the RNA molecule into distinct functional elements: coding regions, self-splicing introns, and terminal structures. This segmentation allows each component to perform its specific function while simplifying the overall design and preparation process, despite the circular topology.
Solution Approach 2:
The patent introduces self-splicing introns as intermediary elements that facilitate the circularization process. These introns act as mediators between the 5' and 3' ends, enabling ligational joining while maintaining the functional integrity of the mRNA molecule.
3Reliability
If Group I self-splicing introns are used for circularization, then circular RNA formation is achieved, but additional elements and processing steps are required
Solution Approach 1:
The patent designs the vector to contain multiple functional elements (coding sequences, self-splicing introns, and terminal structures) that serve multiple purposes. The self-splicing introns simultaneously catalyze their own removal and facilitate circularization, while the terminal structures provide both stability and ligation sites, reducing the need for separate processing steps.
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 extends the duration of protein expression by stabilizing mRNA through circularization, allowing for efficient and prolonged therapeutic effects.
Implementation Method 1
the 5' element and the 3' element form a thermodynamically stable multiple way junction RNA structure
Implementation Method 2
forming a circular RNA that is translatable and/or biologically active inside a cell
Implementation Method 3
a 3' Group I self-splicing intron fragment containing a 3' splice site dinucleotide, d) a 5' Group I self-splicing intron fragment containing a 5' splice site dinucleotide
Implementation Method 4
a 5' element comprising none or at least one stem-loop structure, e) a 3' element comprising none or at least one stem-loop structure
Data Source
AI summary
A method of preparing a circular RNA includes transcribing a vector to form a precursor RNA, in which the vector includes the following elements operably connected to each other and arranged in the following sequence: a) a 5′ element, b) a 3′ Group I self-splicing intron fragment containing a 3′ splice site dinucleotide, c) none or an element containing an internal ribosome entry site (IRES) and a protein coding region or an element containing a noncoding region, d) a 5′ Group I self-splicing intron fragment containing a 5′ splice site dinucleotide, and e) a 3′ element, in which 5′ element and 3′ element form a stable structure with a Gibbs free energy (ΔG) from −190 kcal/mol to −9.0 kcal/mol, provided that the stable structure is not a duplex with at least 95% base pairing between 5′ element and 3′ element, in which 3′ Group I self-splicing intron fragment and 5′ Group I self-splicing intron fragment form a self-cleaving and self-ligating RNA molecule, thereby generating circular RNA.