A comparative exploration of mRNA capping enzymes

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Messenger RNA (mRNA) technology is crucial for medical applications, necessitating enhanced *in vivo* expression efficiency.
• The 5' cap structure of mRNA is vital for protecting it from degradation and initiating translation, making *in vitro* capping a key strategy.
• Optimizing mRNA capping enzymes is essential for advancing mRNA-based therapeutics and vaccines.

Purpose of the Study:

• To identify and characterize novel, highly efficient mRNA capping enzymes from various viral sources.
• To optimize the heterologous expression and solubility of these capping enzymes for practical applications.
• To compare the *in vivo* performance of different viral capping enzymes in human cells.

Main Methods:

• Seven viral mRNA capping enzymes were selected for heterologous expression in eukaryotic and prokaryotic systems.
• Different host cells, including *Escherichia coli*, were evaluated for optimal enzyme expression.
• Soluble labels, such as maltose-binding protein, were screened to enhance enzyme solubility.
• mRNA capping efficiency was assessed *in vitro* and *in vivo* following transfection into human cells.

Main Results:

• *Escherichia coli* was determined to be the most suitable host for expressing the viral capping enzymes.
• Maltose-binding protein demonstrated the best solubility enhancement for the capping enzymes.
• The bluetongue virus capping enzyme achieved the highest mRNA transfection efficiency in human cells.
• This enzyme exhibited 38% greater efficiency compared to the commonly used vaccinia virus capping enzyme.

Conclusions:

• The bluetongue virus capping enzyme represents a significant improvement over existing enzymes for *in vitro* mRNA capping.
• Optimized expression and solubility strategies enhance the utility of viral capping enzymes.
• These findings will accelerate the development and broaden the applications of mRNA technology in medicine.