In Situ Circularized RNA via Ribozyme Ligation
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Solution Overview
Problem
Current methods for producing circular RNAs lack simplicity and scalability, limiting their application in research and translational settings due to instability issues with mRNA therapeutics.
Innovation Solution
A linearized ribozyme activated RNA construct is developed, comprising specific ligation sequences and an IRES sequence, which, when introduced into cells, are ligated by an RNA ligase to form a circular RNA construct, enhancing stability and protein production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If mRNA stability is improved through circularization strategies, then mRNA stability is enhanced, but simple and scalable approaches for efficient in vitro production and purification are lacking
Solution Approach 1:
The patent incorporates ribozyme sequences and ligation stems directly into the linear RNA construct before delivery. These elements are pre-positioned to enable automatic self-circularization upon cellular entry, eliminating the need for complex in vitro circularization procedures while achieving enhanced stability.
Solution Approach 2:
The linear RNA construct contains self-contained elements (ribozymes for cleavage and complementary ligation stems for annealing) that automatically drive the circularization process inside cells without requiring external enzymes or complex purification steps, thus simplifying manufacturing while improving stability.
2Reliability
If circularization strategies are implemented to remove free ends, then exonuclease-mediated degradation is prevented, but scalable production methods remain limited
Solution Approach 1:
The linear RNA is designed with pre-positioned ribozyme sequences and ligation stems that are transcribed together in a single step. This preliminary arrangement allows high-throughput production of linear precursors that automatically convert to stable circular forms, maintaining both productivity and reliability.
3Adaptability or versatility
If linear RNA constructs with ligation sequences are used, then in situ circularization is enabled, but the complexity of the construct design increases
Solution Approach 1:
The patent uses ribozyme sequences as intermediaries that simplify the overall design. These ribozymes perform the cleavage function that would otherwise require complex enzymatic treatments, while their products naturally form ligation stems. This intermediary approach enables circularization capability without proportionally increasing design complexity.
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 approach enables efficient in vitro production and purification of circular RNAs, improving their stability and translational efficiency, thus broadening their applications in protein production systems and gene therapy.
Implementation Method 1
the first and second ligation sequences form a stem substrate for an RNA ligase
Data Source
AI summary
Provided herein are engineered linear RNA polynucleotide molecules that form circular RNA polynucleotide molecules in cells. Also provided are DNA constructs encoding the engineered linear RNA polynucleotide molecules.