Directed evolution of a sequence-specific covalent protein tag for RNA labeling

Efficient methods for conjugating proteins to RNA are needed for RNA delivery, imaging, editing, interactome mapping, as well as for barcoding applications. Non-covalent coupling strategies using viral RNA binding proteins such as MCP have been applied extensively but are limited by tag size, sensitivity, and dissociation over time. We took inspiration from a sequence-specific, covalent protein-DNA conjugation method based on the Rep nickase of a porcine circovirus called “HUH tag”. Though wild-type HUH protein has no detectable activity towards an RNA probe, we engineered an RNA-reactive variant, called rHUH, through 7 generations of yeast display-based directed evolution. Our 13.4 kD rHUH has 12 mutations relative to HUH, and forms a covalent tyrosine-phosphate ester linkage with a 10-nucleotide RNA recognition sequence (“rRS”) within minutes. We engineered the sensitivity down to 1 nM of target RNA, shifted the metal ion requirement from Mn ²⁺ towards Mg ²⁺ , and demonstrated efficient labeling in mammalian cell lysate. This work paves the way toward a new methodology for sequence-specific covalent protein-RNA conjugation in biological systems.