Self-Circularized RNA Structure for Stable Protein Expression
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Solution Overview
Problem
Existing mRNA-based therapeutic agents face challenges with instability and short half-life in vivo, limiting their effectiveness as therapeutic tools.
Innovation Solution
A self-circularized RNA construct is developed through a self-targeting and splicing reaction, featuring a 5' - IGS - ribozyme - gene of interest - target site - 3' structure, which forms a circRNA capable of expressing peptides or proteins efficiently and stably, utilizing a Group I intron ribozyme for trans-splicing and including an IRES region for rapid protein translation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If mRNA is used as a therapeutic agent, then protein expression can be achieved, but the mRNA easily degrades in vivo and has a relatively short half-life
Solution Approach 1:
The patent applies circularization of the RNA structure (forming a circle instead of a linear molecule) to protect the mRNA from degradation. The circular configuration eliminates free ends that are susceptible to enzymatic degradation, thereby improving stability and extending half-life in vivo while maintaining protein expression capability
Solution Approach 2:
The patent employs self-circularization mechanisms where the RNA molecule autonomously forms circular structures through intramolecular reactions. This self-service approach allows the RNA to protect itself from degradation without requiring external intervention, achieving both circularization and stability enhancement
2Reliability
If circular RNA is formed through traditional methods, then stability is improved, but additional GTP treatment is required which increases process complexity
Solution Approach 1:
The patent employs self-circularization mechanisms where the RNA molecule autonomously forms circular structures through intramolecular reactions. This self-service approach allows the RNA to protect itself from degradation without requiring external intervention, achieving both circularization and stability enhancement
Solution Approach 2:
The patent removes the requirement for external GTP treatment and circularization enzymes by incorporating self-circularization sequences directly into the RNA structure. This extraction of the circularization function from external systems integrates the process into the RNA molecule itself, simplifying the overall process
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 self-circularized RNA construct forms circRNA with enhanced stability and long half-life, enabling efficient expression of functional RNAs such as miRNA, mRNA vaccines, and proteins without additional GTP treatment, suitable for in vitro, cellular, and in vivo applications.
Implementation Method 1
the IGS region and the target site form a guanine (G) : uracil (U) wobble base pair
Implementation Method 2
the ribozyme may be a Group I intron ribozyme
Data Source
Figure 1A
Figure 1B
Figure 2A
AI summary
A self-circularized RNA structure of the present invention can be expressed in a DNA vector and, at the same time, form circRNA by being circularized through a self-targeting and splicing reaction, wherein the circRNA consists of only a gene of interest. The gene of interest includes an IRES region, an initiation codon and a termination codon, and thus enables the rapid expression of a peptide and a protein.