HUH Endonuclease-Mediated DNA-Protein Conjugates: Sequence-Specific Tools and Cellular Applications

This Highlight review article summarizes recent advances in employing HUH endonucleases as self-labeling protein tags for the sequence-specific covalent conjugation of unmodified ssDNA and examines their applications in cellular studies via engineered DNA–protein conjugates. We outline the structural basis and catalytic mechanism of the conserved HUH and Y motifs, which enable high selectivity, bioorthogonality, and robust conjugation under physiological conditions. Recent applications demonstrate the versatility of HUH-based DNA–protein conjugates in programmable cellular interface engineering, targeted therapeutic delivery, and enhancement of genome editing systems such as CRISPR–Cas. In the perspective section, we further highlight two emerging directions: computational tools such as the HUHgle platform for predictive substrate design, and directed evolution strategies extending HUH reactivity toward RNA substrates. Together, these advancements establish HUH endonucleases as powerful, programmable tools for generating DNA–protein conjugates that enable innovations in chemical biology, synthetic biology, and therapeutics.