Circular RNA Platform Using Engineered Plasmids for High-Yield Production
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Solution Overview
Problem
Existing methods for producing circular RNA (circRNA) suffer from low yield and efficiency, particularly when larger sized molecules are involved, and often result in the formation of undesired double-stranded stem RNA segments that can trigger immune responses.
Innovation Solution
A novel process involving an engineered parental circular covalently closed synthetic plasmid DNA is used, which includes specific circularization sequences and a bacterial origin of replication, transformed into bacteria to produce circRNA, followed by in vitro transcription and enzymatic or DNAzyme-mediated ligation to achieve high-yield circRNA production without immune-activating stem structures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If existing methods are used to produce circular RNA, then production can be achieved, but yield and efficiency are low
Solution Approach 1:
The patent designs the DNA template with pre-positioned circularization sequences and structural elements (promoters, polyadenylation signals, introns) before transcription. This preliminary configuration enables efficient circular RNA production by eliminating the need for complex post-transcriptional processing and optimization, directly resolving the low yield and efficiency problem
Solution Approach 2:
The patent introduces a DNA template as an intermediary carrier that contains all necessary genetic information and structural elements. This DNA intermediary is transcribed into linear RNA, which then circularizes into the final circRNA product, providing a controlled and efficient production pathway that overcomes direct synthesis limitations
2Reliability
If existing circularization methods are used, then circRNA can be formed, but undesired double-stranded stem segments are created that trigger immune responses
Solution Approach 1:
The patent applies local quality control by designing specific structural elements at different regions of the circRNA: promoters at the 5' end, coding sequences in the middle, and polyadenylation signals at the 3' end. This regional optimization ensures proper folding and prevents formation of immunogenic double-stranded stem segments while maintaining functional integrity
Solution Approach 2:
The patent converts the potential harm of RNA secondary structures into a benefit by deliberately designing the sequence to form controlled stem-loop structures at the ends (through the circularization sequences) that prevent immunogenicity, while ensuring the coding region maintains optimal structure for translation
3Adaptability or versatility
If larger sized circRNA molecules are produced, then more complex proteins can be encoded, but production efficiency decreases significantly
Solution Approach 1:
The patent segments the coding sequence into manageable units within the circRNA structure, using multiple coding sequences separated by appropriate elements. This segmentation allows efficient production of larger circRNA molecules by breaking down the complexity into modular components that can be transcribed and circularized effectively
Solution Approach 2:
The patent transitions from linear to circular topology, adding a dimensional change that allows the RNA to accommodate larger coding capacities. The circular structure provides additional spatial organization that maintains production efficiency even as the coding sequence length increases
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 process yields high amounts of stable circRNA with increased half-life and reduced immunogenicity, enabling efficient protein production and therapeutic applications.
Implementation Method 1
transformed into bacteria to produce circRNA
Implementation Method 2
bacterial origin of replication
Implementation Method 3
followed by in vitro transcription
Implementation Method 4
enzymatic or DNAzyme-mediated ligation to achieve high-yield circRNA production
Data Source
AI summary
The present invention relates to circular RNA platforms, their manufacturing processes from an engineered parental circular covalently closed synthetic plasmid DNA and, uses thereof. Novel engineered circular covalently closed plasmids comprising sequences capable of aiding in RNA circularization either autonomously or when processed with an engineered ligase are provided. The circular RNA platforms of the current invention have improved stability and increased half-life, display exceptional and stable protein production, while avoiding double-stranded intramolecular self-pairing RNA segments.