Circular RNA Production via Self-Cleaving Ribozymes
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
There is a need for effective methods to produce, purify, and utilize synthetic circular polyribonucleotides for therapeutic and engineering applications, as existing methods are inadequate for efficiently generating and utilizing these molecules.
Innovation Solution
A eukaryotic system is employed that includes a polyribonucleotide with specific structural elements such as 5′ and 3′ self-cleaving ribozymes and annealing regions, which, when processed, can be ligated by an RNA ligase to form circular RNA, allowing for the production and isolation of circular RNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If linear polyribonucleotides are produced using conventional methods, then production is simple, but stability and resistance to degradation are poor
Solution Approach 1:
The linear polyribonucleotide is divided into distinct functional segments: a 5' self-cleaving ribozyme, a 5' annealing region, a cargo region, a 3' annealing region, and a 3' self-cleaving ribozyme. This segmentation allows each region to perform its specific function independently, enabling efficient circularization while maintaining production simplicity
Solution Approach 2:
The self-cleaving ribozymes are pre-installed at both ends of the linear polyribonucleotide, and the annealing regions are pre-designed with complementary sequences. When transcribed, these elements automatically catalyze their own circularization without requiring additional processing enzymes or complex purification steps, thereby improving stability without significantly increasing system complexity
2Productivity
If circularization efficiency is increased using advanced methods, then productivity improves, but manufacturing complexity increases
Solution Approach 1:
The self-cleaving ribozymes embedded in the polyribonucleotide sequence automatically catalyze the circularization reaction without requiring external enzymes or complex processing. The complementary annealing regions self-hybridize to bring the 5' and 3' ends together, enabling the molecule to circularize itself with high efficiency and simplicity
Solution Approach 2:
Multiple functions are merged into a single polyribonucleotide sequence: the ribozymes provide both cleavage and ligation capabilities, while the annealing regions provide both structural organization and catalytic facilitation. This merging allows circularization to occur in a single transcription event without requiring 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
This method enables the stable production and isolation of circular RNA, enhancing its stability and utility in therapeutic and engineering applications by promoting efficient circularization and reducing susceptibility to degradation.
Implementation Method 1
wherein: (A) comprises a 5' self-cleaving ribozyme; (E) comprises a 3' self-cleaving ribozyme
Implementation Method 2
The RNA ligase ligates the 5' end and the 3' end of the ligase-compatible linear polyribonucleotide, thereby producing a circular RNA
Implementation Method 3
wherein: (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 eukaryotic system.