Preparation, and enzymatic activity analysis of an engineered capping enzyme

Area of Science:

• Molecular Biology
• Biochemistry
• Virology

Background:

• The vaccinia capping enzyme (VCE) and 2 -O-methyltransferase (VP39) are crucial for synthesizing Cap1 mRNA, essential for mRNA stability, translation, and immune evasion.
• Current enzymatic capping methods utilize VCE and VP39 separately, posing challenges in process complexity and cost.

Purpose of the Study:

• To engineer a fusion capping enzyme (DDGSV) by linking VCE and VP39 to improve mRNA capping efficiency and reduce production costs.
• To assess the structural integrity, expression, and functional performance of the engineered DDGSV enzyme.

Main Methods:

• Constructed a fusion enzyme (DDGSV) by genetically linking VCE and VP39 with a flexible linker.
• Predicted the tertiary structure of DDGSV using AlphaFold2.
• Expressed DDGSV in Escherichia coli and evaluated its capping efficiency and mRNA integrity.
• Tested the performance of DDGSV-capped mRNA in HEK 293T cells.

Main Results:

• AlphaFold2 prediction showed no steric hindrance in the DDGSV fusion enzyme's active sites.
• The engineered DDGSV enzyme achieved 80.19% capping efficiency within 2 hours, comparable to commercial enzymes.
• DDGSV-capped GFP mRNA demonstrated high expression levels in HEK 293T cells.
• RNA dot blotting was explored for rapid efficiency detection, though quantitative methods remain necessary.

Conclusions:

• The engineered DDGSV fusion enzyme effectively synthesizes Cap1 mRNA, offering a promising alternative to current methods.
• DDGSV provides a cost-effective and streamlined approach for mRNA capping, with significant implications for mRNA vaccine development.