Sequence-directed covalent protein–RNA linkages in a single step using engineered HUH-tags

Replication-initiating HUH-endonucleases (reps) are enzymes that form covalent bonds with single-stranded DNA (ssDNA) in a sequence-specific manner to initiate rolling circle replication in plasmids and viruses. These nucleases have been co-opted for use in biotechnology as sequence-directed protein-ssDNA bioconjugation fusion partners dubbed 'HUH-tags'. Here, we describe the engineering and in vitro characterization of a series of laboratory evolved HUH-tag derivatives of PCV2 called E1 and E2. E2 is capable of forming robust covalent bonds with unmodified RNA substrates in a sequence-specific manner. We show that promiscuous rep-RNA interaction can be enhanced through directed evolution from nearly undetectable levels in wildtype enzymes to robust reactivity in final engineered iterations. Subsequent in vitro characterization revealed that engineered enzymes have dramatically increased activity on both cognate ssDNA and noncognate ssRNA substrates. We benchmark our engineered proteins against an engineered variant called rHUH described in recent work from another laboratory pursuing the same function, and we perform an extensive analysis of sequence specificity across a range of metal ion concentrations. Together, these results establish a new class of RNA-reactive HUH-tags that expand the biochemical repertoire of this enzyme family and provide a promising platform for site-specific protein-RNA covalent bioconjugation. This technology has the potential to unlock diverse new applications in biotechnology and molecular engineering.