poly A high fidelity solution

Background

In eukaryotic organisms, poly A tail exists in almost every mRNA. The 3' end poly A tail plays a crucial role in the stability and translation of mRNA. It can prevent the degradation of 3’ end of mRNA by exonuclease. At the same time, it can bind PABPC, and then PABPC interact with eIF4G to form a "closed loop" structure, which helps to stimulate translation initiation. These functions are closely related to poly A tail, so the sequence design of poly A tail is one of the key factors for the quality assurance of mRNA products.

blog
blog

Problems

poly A tail is easily lost during fermentation, which leads to the shortening of poly A tail in mRNA products and the decrease of protein expression efficiency.

Techniques

The strain modification improves the stability of poly A tail in the fermentation process.

The design optimization improves the stability of the poly A tail.

Optimization of the fermentation and purification processes further improves the retention rate of poly A tail, plasmid yield, and purity.

blog

Result

1.The self-developed genetically modified strain RHS2-2 is more capable of maintaining the stability of poly A tail in the subculture process than the commercial strains DH5α and RHS2.

2.Enobio’s patented poly A tail sequence (poly A2.0) has higher stability and comparable protein expression efficiency compared with commercial sequences.

3.The optimized fermentation process reduced the final recombination rate of the fermentation broth in the primary batch to less than 10%. Combined with the optimized plasmid purification and mRNA purification processes, the final mRNA poly A distribution was highly concentrated in the design region, achieving stable and excellent results.

blog
blog
blog
Strain Recombination rate of poly A120 (Passage 1) Recombination rate of poly A120 (Passage 20) Recombination rate of poly A2.0 (Passage 1)
DH5α 72.2% - -
RHS2-2 12.5% 0 10.4%
RHS2 49.0% 0 16.7%

Conclusion

The stability of the poly A region of the template plasmid during fermentation was significantly improved by optimizing the poly A sequence design and modifying the fermentation strain; at the same time, by optimizing the fermentation and purification processes, impurities with incomplete poly A produced during in vitro transcription could be removed, further ensuring the fidelity of poly A in mRNA products.