Regarding the batch preparation of RNA precursors, a commonly used method is in vitro transcription (IVT). The IVT reaction uses linearized plasmid DNA containing the T7 promoter as a template and synthesizes an RNA precursor with nucleoside triphosphates (NTPs) as a substrate in the presence of T7 RNA polymerase.
In vitro cyclization methods include chemical ligation, enzymatic ligation and permuted intron-exon (PIE) method. Chemical ligation and enzymatic ligation are suitable for the cyclization of shorter RNAs. When the fragment is larger than 100 nt, the cyclization rate will greatly decrease. In contrast, the nuclease method based on the PIE system can achieve cyclization of 8 kb sequences.
Under the catalysis of guanosine triphosphate (GTP), the PIE structure undergoes cyclization of extra-intron sequences. Combined with a reasonable strategy to increase the cyclization rate, Yaohai Bio-Pharma can achieve cyclization of up to 4 kb sequences, with a cyclization efficiency of more than 80%.
The in vitro cyclization reaction of RNA flows as follows:
The RNA precursor is synthesized by in vitro transcription, and the PIE component completes self-splicing under the catalysis of GTP to form circRNA.
Services Details
| Process |
Service Details |
Delivery Period (Workday) |
| In vitro transcription and circulation |
Reaction system confirmation |
1-2 |
| In vitro transcription and cyclization reactions |
| RNase R digestion |
| Reaction optimization |
Reaction composition, time optimization |
2-5 |
Our Features
- Rigorous test design and optimization
Up to 4 kb of RNA cyclization can be achieved.
- High cyclization efficiency
A cyclization rate of more than 80% can be achieved through a rational sequence optimization strategy.
- Stringent control of RNase
Through stringent control of RNases in the experimental environment and consumables, RNA degradation is effectively prevented.
Case study
In vitro cyclization and enrichment of circRNA
Based on the PIE system, Yaohai Bio-Pharma has optimized the sequence of circRNA, with a cyclization rate of more than 80% by agarose gel electrophoresis (AGE). Using RNase R to enrich circRNA, E-gel electrophoresis results show that the linear RNA precursor is digested, and after further purification, most of the nicked circRNA can be removed. The purification solution is self-developed by Yaohai Bio-Pharma.
In vitro circulation and enrichment of circRNA
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