Towards programmable metallonucleases via genetic code expansion
The rapid advancement of protein engineering and genetic code expansion technologies over the last decade has reshaped how researchers rationally design proteins with novel catalytic functions. Among these approaches, the site-specific incorporation of unnatural amino acids has enabled the introduction of chemical functionalities that are inaccessible to the canonical amino acid space. In this perspective, we highlight the metal-chelating UAA (2,2′-bipyridin-5-yl) alanine (BpyAla) and its emerging utility in mediating nucleic acid cleavage. Multiple studies have demonstrated the successful site-specific incorporation of BpyAla into proteins of interest, where subsequent metal coordination enables catalytic cleavage of DNA and RNA substrates. Here, we discuss the potential of BpyAla-mediated nucleic acid cleavage, with emphasis on the development of next-generation BpyAla analogues, the exploration of alternative metal cofactors, cooperative and multi-residue design strategies, and the expansion of compatible protein scaffolds and nucleic acid substrates. Designable BpyAla-engineered systems represent an emerging frontier in artificial metallonuclease design, with potential long-term relevance to targeted nucleic acid therapeutics.