Molecular Basis for Rep HUH‐endonuclease Mediated Protein‐DNA Bioconjugation

Self‐labeling enzymes such as the SNAP‐tag, CLIP‐tag, and HALO‐tag are popular systems for specific protein‐DNA bioconjugation. While these fusion tags are useful and efficient, the limited number of orthogonal or non‐cross reactive self‐labeling enzymes pose a bottleneck for development of multiplexed applications reliant on protein‐DNA linkages, such as protein barcoding. We are addressing this bottleneck by exploiting the single‐stranded DNA (ssDNA) processing mechanism of HUH‐endonucleases, so named for a histidine‐hydrophobic residue‐histidine (HUH) motif, to create site‐specific and covalent linkages with unmodified DNA. Several recently developed applications in the fields of gene editing, cell targeting, live cell imaging, among others, are reliant on these so called HUH‐tags. The most efficient and versatile HUH‐tags are 11‐16 kDa replication initiator proteins (Reps), which initiate rolling circle replication in a variety of organisms. We characterized the underlying molecular basis by which Reps recognize ssDNA sequences using structural analysis of co‐crystal structures and by profiling the ssDNA specificity of a panel of Reps using a novel deep sequencing‐based cleavage assay we developed termed HUH‐seq. Combining the information from these results, we swapped residues between two different Reps to create a chimeric Rep with predictably altered specificity. These results are a first step in the direction of creating an HUH‐tag toolkit that may spawn the development of new biotechnologies.