HUH Endonuclease: A Sequence-specific Fusion Protein Tag for Precise DNA-Protein Conjugation

Synthetic DNA-protein conjugates have found widespread applications in diagnostics and therapeutics, prompting a growing interest in developing chemical biology methodologies for the precise and site-specific preparation of covalent DNA-protein conjugates. In this review article, we concentrate on techniques to achieve precise control over the structural and site-specific aspects of DNA-protein conjugates. We summarize conventional methods involving unnatural amino acids and self-labeling proteins, accompanied by a discussion of their potential limitations. Our primary focus is on introducing HUH endonuclease as a novel generation of fusion protein tags for DNA-protein conjugate preparation. The detailed conjugation mechanisms and structures of representative endonucleases are surveyed, showcasing their advantages as fusion protein tag in sequence selectivity, biological orthogonality, and no requirement for DNA modification. Additionally, we present the burgeoning applications of HUH-tag-based DNA-protein conjugates in protein assembly, biosensing, and gene editing. Furthermore, we delve into the future research directions of the HUH-tag, highlighting its significant potential for applications in the biomedical and DNA nanotechnology fields.