pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A) Sequences

Area of Science:

• Molecular Biology
• Biotechnology
• Synthetic Biology

Background:

• Demand for large-scale in vitro transcribed (IVT) mRNA synthesis is rising due to its use in cell engineering and therapeutics.
• The 3' polyadenosine (poly(A)) tail length is crucial for IVT mRNA potency and translational efficiency.
• Current methods limit poly(A) tail lengths to approximately 120 base pairs (bp) due to homopolymeric tract instability in circular plasmids or PCR products.

Purpose of the Study:

• To develop a novel method for generating extended poly(A) tracts for IVT mRNA production.
• To overcome limitations of current methods in producing long and stable poly(A) tails.
• To create a plasmid system for consistent and precisely defined IVT mRNA poly(A) tails and 3' termini.

Main Methods:

• Utilized a linear plasmid system (pJazz) to propagate extended poly(A) tracts.
• Modified the pJazz plasmid to create a new system (pEVL) with a T7 promoter, extended multiple cloning site, and a type-IIS restriction site.
• The type-IIS restriction site facilitates the generation of IVT mRNA with defined poly(A) tracts and 3' termini.

Main Results:

• Linear plasmids successfully propagated poly(A) tracts up to approximately 500 bp for IVT mRNA production.
• The modified plasmid, pEVL, enables the generation of IVT mRNA with consistent and defined poly(A) tail lengths.
• The system allows for precise control over the 3' termini of the synthesized mRNA.

Conclusions:

• The developed pEVL plasmid system is effective for producing IVT mRNA with extended and precisely defined poly(A) tails.
• This method overcomes previous limitations, enabling the synthesis of more potent mRNA for various applications.
• The ability to generate consistent, defined mRNA constructs is critical for advancing mRNA-based technologies.