Scarless RNA Circularization Using Group I Intron Ribozymes
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Solution Overview
Problem
Current methods for RNA circularization, such as the permuted intron-exon (PIE) system, are complex, inefficient, and introduce extraneous fragments, leading to immune responses and complications in production scale-up.
Innovation Solution
A novel RNA construct design using a group I intron-based circularization system with an intact ribozyme core, featuring specific recognizer sequences and a ribozyme core sequence, allows for self-circularization without extraneous fragments, ensuring high efficiency and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the permuted intron-exon (PIE) system is used for RNA circularization, then circularization can be achieved, but the system is complex and introduces extraneous fragments
Solution Approach 1:
The patent extracts and removes the extraneous fragments (P1, P2, P3, P4 sequences) from the circularization system, retaining only the essential ribozyme core sequences (P5-P8) needed for circularization. This extraction eliminates the source of immune responses and simplifies the system while maintaining circularization functionality.
Solution Approach 2:
The patent converts the previously harmful extraneous fragments into beneficial nothingness by deleting them entirely. The ribozyme core sequences that were part of the PIE system are now used in a purified form without the harmful P1-P4 sequences, transforming a harmful system into a clean, efficient circularization method.
2Reliability
If the permuted intron-exon (PIE) system is used for RNA circularization, then circularization can be achieved, but extraneous fragments lead to immune responses
Solution Approach 1:
The patent extracts and removes the extraneous fragments (P1, P2, P3, P4 sequences) from the circularization system, retaining only the essential ribozyme core sequences (P5-P8) needed for circularization. This extraction eliminates the source of immune responses and simplifies the system while maintaining circularization functionality.
3Reliability
If enzyme ligation-mediated circularization is used, then circularization can be achieved, but the process is inefficient and produces intermolecular end-joining by-products
Solution Approach 1:
The patent replaces the mechanical enzyme ligation process with a chemical ribozyme-catalyzed transesterification reaction. The ribozyme core sequences (P5-P8) catalyze the direct formation of phosphodiester bonds between RNA ends through chemical mechanism, eliminating the need for protein enzymes and their associated by-products.
Solution Approach 2:
The ribozyme core sequences perform self-circularization through catalytic transesterification reactions, where the RNA molecule itself (via the ribozyme core) catalyzes its own circularization without requiring external enzyme ligases. This self-service mechanism eliminates intermolecular end-joining by-products.
4Reliability
If plasmid vectors are used for in vivo circularization, then circularization can be achieved, but delivery is difficult and genomic integration risk exists
Solution Approach 1:
The patent extracts the circularization function from plasmid vectors and delivers it as a standalone RNA construct with ribozyme core sequences. This eliminates the need for complex plasmid delivery systems and reduces the risk of genomic integration, as only the essential circularization sequences are delivered rather than entire plasmid vectors.
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 novel RNA construct achieves efficient and scarless circularization of nucleotide sequences, reducing immune response risks and simplifying production processes, while maintaining the advantages of circular RNAs like stability and low immunogenicity.
Implementation Method 1
a ribozyme core sequence operably linked to an internal guide sequence (IGS), wherein the ribozyme core sequence encodes a ribozyme core having the catalytic activity of a group I intron ribozyme
Implementation Method 2
The group I introns are naturally occurring cis-splicing ribozymes that can splice an RNA transcript and remove themselves from the primary transcript by autocatalyzing two consecutive trans-esterification reactions and joining the two flanking exons
Implementation Method 3
the 5' end nucleotide of the IGS and the 3' end nucleotide of the target site form a non-Watson-Crick base pair to define a 5' splice site
Implementation Method 4
R1 and R2 are positioned at opposite ends of the RNA construct, such that hybridization of the first and second pairing sequences results in the formation of a duplex-containing structure to define a 3' splice site
Data Source
AI summary
The disclosure relates to novel RNA ribozyme constructs encoding foreign proteins or functional RNAs, with a circularization system based on group I introns, which are capable of self-circularizing with high efficiency without introducing extraneous fragments, as well as to methods of using the constructs to make circular RNAs.