Enhancing mRNA stability and translational potential through tailored modifications at the 3' end

Area of Science:

• Molecular and Cellular Biology
• Biochemistry
• Synthetic Biology

Background:

• Poly(A) tails on messenger RNA (mRNA) are crucial for regulating mRNA stability and translation.
• Deadenylation, the shortening of the poly(A) tail, is the initial step in major mRNA decay pathways.
• Protecting the 3' end of synthetic mRNA is vital for enhancing its therapeutic potential.

Purpose of the Study:

• To develop a post-transcriptional strategy for protecting the 3' end of synthetic mRNA.
• To investigate the use of enzymatically ligated, chemically modified dinucleotides for mRNA stabilization.
• To assess the impact of 3'-end modification on mRNA deadenylation and translation efficiency.

Main Methods:

• Enzymatic ligation of 5'-phosphorylated dinucleotides (2'-O-methyl and/or phosphorothioate modified) to model oligoadenylates and synthetic Gaussia luciferase (GLuc) mRNA using T4 RNA ligase 1.
• In vitro deadenylation assays using human CNOT7 enzyme.
• In vitro translation assays in rabbit reticulocyte lysate.
• In vivo protein expression analysis in mammalian cell lines (A549, JAWSII, HEK293).

Main Results:

• Ligated dinucleotide-modified mRNAs demonstrated significant resistance to CNOT7-mediated deadenylation in vitro compared to unmodified controls.
• 3'-end modified GLuc mRNAs exhibited comparable translation efficiency in rabbit reticulocyte lysate to unmodified mRNA.
• In mammalian cells, specific adenosine dinucleotide modifications (pApA_m and pAp_sA_m) enhanced cumulative GLuc production by up to 163% (HEK293) and 79% (A549).

Conclusions:

• Dinucleotide ligation is an effective and modular strategy for engineering the 3' end of synthetic mRNA.
• This approach enhances mRNA resistance to deadenylation, a key factor in mRNA decay.
• 3'-end modification via dinucleotide ligation can significantly improve the translational performance of therapeutic mRNA candidates.