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Updated: Jan 20, 2026
Cis-acting Elements in mRNA stability and Gene Expression
Poly(A) variants supporting robust transmission stability in bacteria and high protein expression in animals for mRNA
Abstract:
Poly(A) tail is crucial in regulating mRNA stability and protein translation. Thus, it is an essential element of mRNAs transcribed in vitro for mRNA medicines. However, the repetitive nature of the poly(A) tail can lead to significant poly(A) length variation and compromise the quality of the mRNA drug substance. Previous studies improved poly(A) transmission stability by inserting a non-adenosine spacer. Here, we designed new segmented poly(A) variants and evaluated their transmission stability in bacteria transformation and their ability in supporting protein expression in animals. We identified specific variants with multiple non-adenosine insertions that can maintain high transmission stability with robustness in Escherichia coli and facilitate high protein expression in animals. Among the newly designed poly(A) variants, RG2 showed high and consistent transmission stability comparable to the A30-70 variant that is the industry gold standard but higher protein expression in animals than A30-70. We also isolate new factors that can influence the stable transmission of poly(A), such as poly(A) surrounding sequences and bacteria culture temperature. Thus, our work offers new tools valuable for rapidly developing mRNA vaccines and therapeutics.
Insights
New poly(A) tail variants improve mRNA stability and protein expression for vaccines and therapeutics. These segmented poly(A) sequences offer robust bacterial transmission and enhanced animal expression, advancing mRNA medicine development.
Area of Science:
- Biotechnology
- Molecular Biology
- mRNA Therapeutics
Background:
- The poly(A) tail is vital for mRNA stability and translation, crucial for *in vitro* transcribed mRNA medicines.
- Repetitive poly(A) sequences can cause length variations, impacting mRNA drug substance quality and consistency.
Purpose of the Study:
- To design and evaluate novel segmented poly(A) variants for improved mRNA transmission stability and protein expression.
- To identify factors influencing stable poly(A) transmission in bacterial systems.
Main Methods:
- Design of segmented poly(A) variants with non-adenosine insertions.
- Evaluation of transmission stability in *Escherichia coli* transformation.
- Assessment of protein expression levels in animal models.
Main Results:
- Specific segmented poly(A) variants demonstrated robust transmission stability in bacteria.
- The RG2 variant showed comparable transmission stability to the industry standard (A30-70) but superior protein expression in animals.
- Identified poly(A) surrounding sequences and bacterial culture temperature as factors affecting stable poly(A) transmission.
Conclusions:
- Novel segmented poly(A) variants offer enhanced stability and protein expression for mRNA therapeutics.
- The RG2 variant presents a promising alternative to current standards for mRNA medicine development.
- Understanding factors influencing poly(A) transmission can optimize mRNA production and application.
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