Circular RNA Production via Self-Cleaving Ribozymes
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Solution Overview
Problem
There is a need for methods to produce, purify, and utilize circular polyribonucleotides, which are ubiquitously expressed in human tissues but require efficient production and application techniques.
Innovation Solution
A prokaryotic system is used to circularize linear polyribonucleotides by incorporating 5′ and 3′ self-cleaving ribozymes and annealing regions, which are then ligated by an RNA ligase to produce circular RNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If linear polyribonucleotides are used as starting material, then the production process is simple, but the circularization efficiency is low
Solution Approach 1:
The linear polyribonucleotide is divided into functional segments: 5' self-cleaving ribozyme, 5' annealing region, cargo, 3' annealing region, and 3' self-cleaving ribozyme. This segmentation enables efficient circularization through controlled cleavage and ligation at specific sites while maintaining simplicity in the overall process.
Solution Approach 2:
The 5' and 3' self-cleaving ribozymes perform the cleavage function autonomously without requiring external enzymatic intervention. This self-service mechanism simplifies the production process by eliminating the need for additional cleavage enzymes while achieving efficient circularization.
2Productivity
If prokaryotic system is used, then the production scalability is high, but the RNA stability may be reduced
Solution Approach 1:
The circular structure itself acts as a protective cushion against degradation by eliminating free ends that are typically targeted by nucleases. Additionally, the annealing regions provide structural stability that cushions the cargo against environmental factors in the prokaryotic system.
Solution Approach 2:
The patent modifies the RNA structure by adding specific functional elements (ribozymes, annealing regions) that change the physical and chemical parameters of the RNA molecule, making it more stable in the prokaryotic environment while maintaining scalability.
3Manufacturing precision
If self-cleaving ribozymes are incorporated, then the circularization specificity is high, but the construct complexity increases
Solution Approach 1:
The self-cleaving ribozyme sequences serve multiple functions: they define the circularization sites, provide structural anchors for the annealing regions, and enable controlled cleavage. This multi-functionality achieves high circularization specificity while minimizing the overall construct complexity.
Solution Approach 2:
The annealing regions act as intermediaries that facilitate the interaction between the 5' and 3' ends of the polyribonucleotide. These intermediary elements enable specific circularization without requiring direct complex interactions between the ribozyme sites themselves.
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
This method enables the efficient production and purification of circular RNA, enhancing its stability and utility in therapeutic and engineering applications.
Implementation Method 1
A) comprises a 5' self-cleaving ribozyme; (E) comprises a 3' self-cleaving ribozyme; cleavage of the 5' self-cleaving ribozyme and of the 3' self-cleaving ribozyme produces a ligase-compatible linear polyribonucleotide
Implementation Method 2
cleavage of the 5' self-cleaving ribozyme and of the 3' self-cleaving ribozyme produces a ligase-compatible linear polyribonucleotide
Implementation Method 3
the RNA ligase ligates the 5' end and the 3' end of the ligase-compatible linear polyribonucleotide, thereby producing a circular RNA
Implementation Method 4
B) comprises a 5' annealing region; (D) comprises a 3' annealing region
Data Source
AI summary
The present disclosure relates, generally, to methods for producing, purifying, and using circular RNA from a prokaryotic system.