Large-scale analysis of mRNA sequences localized near the start and amber codons and their impact on the diversity of mRNA display libraries

Extremely diverse libraries are essential for effectively selecting functional peptides or proteins, and mRNA display technology is a powerful tool for generating such libraries with high diversity. Particularly, the protein-puromycin linker (PuL)/mRNA complex formation yield is determining for preparing the libraries. However, how mRNA sequences affect the complex formation yield remains unclear. To study the effects of N-terminal and C-terminal coding sequences on the complex formation yield, puromycin-attached mRNAs containing three random codons after the start codon (32,768 sequences) or seven random bases next to the amber codon (6,480 sequences) were translated. The present study provides insights into how mRNA sequences affect the protein/mRNA complex formation yield and will accelerate the identification of functional peptides and proteins involved in various biological processes and having therapeutic applications.