Dumbbell Circular RNA Precursors for DNA-Splint-Free Ligation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for preparing circular RNA (circRNA) using T4 RNA ligase 2 are inefficient and require DNA splints, which complicate the isolation and purification process, and circRNA produced for protein expression is large in size, limiting its effectiveness.
Innovation Solution
A circular RNA precursor with a dumbbell structure that self-hybridizes at its ends, allowing for efficient ligation by T4 RNA ligase 2 without a DNA splint, and includes a 3'DB region of 10 to 29 nt and a 5'DB region of 3 to 15 nt, with a total length of 30 bp or less, and can incorporate genes up to 5000 nt for protein expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA splints are used to prepare circRNA using T4 RNA ligase 2, then the ligation can be achieved, but the isolation and purification process becomes complicated
Solution Approach 1:
The invention extracts and removes the DNA splint component from the circRNA preparation system. By designing a dumbbell structure that self-hybridizes through complementary sequences, the patent eliminates the need for external DNA splints, thereby simplifying the isolation and purification process while maintaining ligation efficiency
Solution Approach 2:
The dumbbell structure incorporates self-complementary sequences that automatically hybridize to form a stable structure without requiring external DNA splints. This self-service mechanism allows the RNA molecule to prepare itself for ligation, eliminating the need for additional purification steps to remove DNA components
2Adaptability or versatility
If circRNA is produced for protein expression, then the functional application is achieved, but the size becomes large which limits effectiveness
Solution Approach 1:
The invention applies local quality by concentrating the essential functional elements within a compact dumbbell structure. The self-complementary sequences are strategically positioned to provide the necessary stability and ligation efficiency without adding excessive length, allowing the circRNA to maintain protein expression capability while minimizing size
Solution Approach 2:
The circRNA is segmented into functional domains: the dumbbell structure for stability and ligation, and the gene of interest for protein expression. This segmentation allows the non-coding regions to be minimized while preserving the essential functions, thereby reducing overall size without compromising protein expression capability
3Reliability
If traditional methods are used to prepare circRNA, then the basic structure is achieved, but the preparation efficiency is low
Solution Approach 1:
The dumbbell structure is designed in advance with self-complementary sequences that pre-form a stable hybridized structure. This preliminary action of self-hybridization creates an optimal substrate for T4 RNA ligase 2, significantly enhancing ligation efficiency while maintaining structural integrity of the final circRNA product
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 method achieves higher efficiency in preparing circRNA, reduces the need for additional RNA removal steps, and allows for the production of stable circRNA suitable for gene therapy by minimizing immune response induction, with improved ligation efficiency and yield.
Implementation Method 1
the 3'DB AS region consists of a reverse complementary sequence to the 3'DB SS region, the 3'DB AS region and the 3'DB SS region form base pairs
Implementation Method 2
T4 RNA ligase 2 (Rnl2) is an enzyme for linking a 3'-end and a 5'-end of double-stranded RNA or double-stranded RNA/DNA hybrid
Data Source
Figure 1
Figure 2A
Figure 2B
AI summary
The present disclosure relates to a circular RNA precursor having a dumbbell-shaped secondary structure, and the like. According to the present disclosure, the circular RNA precursor uses T4 RNA ligase 2 (Rnl2), but does not require a DNA splint in preparing circular RNA, and furthermore, it is possible to prepare circular RNA with greater efficiency than using the DNA splint. In addition, in preparation of circular RNA using a ribozyme, the ribozyme needs to be finally removed, but in the present disclosure, an additional RNA removal step is not required. In addition, the circular RNA precursor of the present disclosure may be prepared as circular RNA containing a protein-encoding gene as GOI, and thus can be used as a gene therapy for the purpose of protein expression.