'Head-to-Head' mRNA display for the translation of multi-copied proteins with a free C-terminus

With the development of various methods for affinity-based selection of proteins such as phage display, ribosomal display, and mRNA display, the progress in this field has been gradually shifting to function-based selection, such as through single-molecule observation, genetic selection, and compartmentalization technologies. In this vein, we present an opposite link mode of mRNA display termed as a 'Head-to-Head' (H2H) link. The key technique in H2H, formation of a covalent bond between O⁶-benzylguanine (BG) and O⁶-alkylguanine-DNA alkyltransferase (AGT), was demonstrated to be workable in H2H ligation, where mRNA is linked to a nascent AGT via a BG-DNA linker, resulting in a "(C-terminus) protein-BG-DNA linker-mRNA (5'-terminus)" conjugate. Thus, a head (N-terminus) to head (5'-terminus) linkage is formed. Among the advantages of H2H, the generation of multi-copied proteins is the most promising and was proven to be possible owing to the restored stop codon, which had been intentionally removed in the conventional mRNA display. Another advantage is obviously having a free C-terminus of the protein, which can be used for modifications such as C-terminal methylation, α-amidation, and others, which occur in nature. A superior merit of H2H is that it makes it possible to use a single construct commonly in mRNA display (affinity-based) and compartmentalization technologies (function-based) without requiring complicated construct changes.