Self-Circularized RNA via Group I Self-Splicing for Longer 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.
Innovation Solution
The method involves transcribing a vector to form a precursor RNA with specific elements arranged to form a thermodynamically stable multiple way junction RNA structure, which can be circularized to create a biologically active circular RNA capable of extended protein 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 and is rapidly degraded by exonucleases
Solution Approach 1:
The patent applies curvature transformation by converting linear mRNA into circular RNA (circRNA) structure. The circular topology eliminates the 5' and 3' ends that are recognized by exonucleases, thereby preventing degradation while maintaining synthesis capability through in vitro transcription methods.
Solution Approach 2:
The circular RNA structure is self-protecting against exonuclease degradation due to its continuous circular topology. The molecule serves its own protection function by lacking terminal structures that would otherwise be vulnerable to enzymatic attack, extending its half-life without requiring additional protective modifications.
2Duration of action of stationary object
If circular RNA is synthesized through traditional ligation methods, then half-life is extended, but the process becomes complex and requires additional enzymatic steps
Solution Approach 1:
The patent extracts and eliminates the need for external ligation enzymes by incorporating self-splicing intron elements directly into the RNA sequence. The Group I intron structure performs the circularization function internally through its own catalytic activity, removing the requirement for separate ligase enzymes and simplifying the overall process.
Solution Approach 2:
The Group I intron element acts as a self-catalyzing module that performs the circularization reaction without requiring external enzymatic assistance. The intron's ribozyme activity enables the RNA molecule to circularize itself, reducing process complexity while achieving the desired half-life extension.
3Productivity
If Group I intron elements are used for self-circularization, then circularization efficiency increases, but the RNA sequence design becomes more complex
Solution Approach 1:
The patent segments the RNA molecule into functional modules: 5' element, Group I intron, coding region, and 3' element. This modular design allows the complex circularization function to be distributed across separate components, making the sequence design more manageable while maintaining high circularization efficiency through the catalytic intron element.
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 circular RNA exhibits enhanced stability and prolonged protein expression, overcoming the limitations of linear mRNA degradation and extending its duration in biological systems.
Implementation Method 1
the 5′ element and the 3′ element form a thermodynamically stable multiple way junction RNA structure
Implementation Method 2
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
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 the 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.