Single-Pot circRNA Production via Group I Intron Self-Splicing
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Solution Overview
Problem
Current methods for producing circular RNAs (circRNAs) are inefficient and costly, requiring multiple reaction and purification steps, which are time-consuming and may introduce contaminants.
Innovation Solution
A single-pot in vitro transcription reaction method is used to produce circRNAs from a DNA construct encoding a linear RNA precursor, utilizing an RNA polymerase and nucleotides under conditions that allow transcription and circularization of the linear RNA precursor through activation of Group I intron fragments, without the need for additional reagents or steps like DNase I treatment or separate GTP treatment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple reaction and purification steps are used to produce circRNAs, then the production can be completed, but the process becomes time-consuming and introduces contaminants
Solution Approach 1:
The patent combines transcription and circularization reactions into a single-pot in vitro transcription system. The DNA construct containing Group I intron sequences is transcribed by RNA polymerase into linear RNA precursor, which then automatically circularizes through intron-mediated splicing in the same reaction mixture without intermediate purification steps.
Solution Approach 2:
The DNA construct is designed in advance with Group I intron sequences flanking the target RNA region. These introns are positioned to enable automatic circularization of the transcribed RNA product without requiring additional treatment steps, as the circularization capability is built into the template structure beforehand.
2Reliability
If multiple reaction and purification steps are used to produce circRNAs, then the production can be completed, but the process becomes complex and costly
Solution Approach 1:
The patent merges transcription and circularization into a single reaction system. The reagent composition contains RNA polymerase and nucleotides that simultaneously support DNA template transcription and intron-mediated RNA circularization, eliminating the need for separate reaction vessels and purification operations.
Solution Approach 2:
The Group I intron sequences serve multiple functions: they are transcribed as part of the linear RNA precursor and simultaneously act as self-splicing elements that catalyze circularization. This multi-functionality is built into the DNA construct design, reducing the need for additional reagents or enzymes.
3Reliability
If DNase I treatment and separate GTP treatment steps are used, then circularization can be achieved, but additional reagents and steps are required
Solution Approach 1:
The Group I intron sequences possess self-splicing activity that enables automatic circularization of the RNA precursor without requiring external enzymes or additional reagents like DNase I or supplemental GTP. The introns catalyze their own excision and the ligation of flanking exons to form the circular RNA structure.
Solution Approach 2:
The Group I intron sequences act as self-contained intermediaries that mediate the circularization process. During transcription, these introns are incorporated into the linear RNA and then automatically excise themselves through ribozyme activity, facilitating exon ligation and circle formation without requiring separate treatment 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 simplifies the production of circRNAs by reducing the number of reaction steps and minimizing potential contaminants, thereby saving time and resources while maintaining the stability and effectiveness of the circRNAs.
Implementation Method 1
contacting the DNA construct with a reagent composition comprising an RNA polymerase, adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP), guanosine 5'-triphosphate (GTP) and cytosine 5'-triphosphate (CTP) under conditions that allow transcription of the DNA construct into the linear RNA precursor
Implementation Method 2
the circularization comprises activation of the 3' catalytic Group I intron fragment and the 5' catalytic Group I intron fragment to splice the 3' exon sequence and the 5' exon sequence from the linear RNA precursor
Data Source
AI summary
The present application provides methods for producing circular RNAs (circRNAs) from a DNA construct encoding a linear RNA precursor, wherein the linear RNA precursor comprises from the 5′-end to the 3′ end: a 3′ catalytic Group I intron fragment, a 3′ exon sequence, an effector RNA sequence, a 5′ exon sequence, and a 5′ catalytic Group I intron fragment, wherein the method comprises an in vitro single-pot reaction. In some embodiments, the single-pot reaction does not comprise supplementing the reagent composition with GTP, a divalent metal ion such as Mg2+, or DNase I prior to circularization of a linear RNA precursor.